Embryonic stem cell specific micrornas promote induced pluripotency

ABSTRACT

The methods of the present application describe that introduction of physiologically relevant miRNAs can enhance or modulate somatic cell reprogramming, generating induced pluripotent stem cells (iPS cells). These miRNAs did not further enhance reprogramming in the presence of cMyc. Furthermore, unlike previously described methods of generating iPS cells, such as through the introduction of genetic elements using viruses, the methods of the present invention reduce the risk of activating oncogenes in the iPS cells. The methods of the invention generate iPS cells that can be free of genetic modifications and thus have greater potential for use as therapeutic agents than those generated by existing methods.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority benefit of U.S. Provisional ApplicationSer. No. 61/165,865 filed on Apr. 1, 2009, the contents of which areincorporated herein by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under NIH grants K08NS48118 and RO1 NS057221 awarded by the National Institutes of Health(The Government of the United States of America as represented by theSecretary of the Department of Health and Human Services); thegovernment has certain rights in the invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

NOT APPLICABLE

FIELD OF THE INVENTION

The present invention relates to development of a method for makinginduced pluripotent stem (iPS) cells by introducing microRNAs (miRNAs)into somatic cells. In some embodiments, the somatic cells are eitheradult or embryonic, mouse or human, fibroblasts. Unlike previouslydescribed methods of generating iPS cells, such as through theintroduction of genetic elements using viruses, the methods of thepresent invention reduce the risk of activating oncogenes in the iPScells. The methods of the invention can create iPS cells that lack atleast one genetic modification, or are free of genetic modifications,and thus have greater potential for use as therapeutic agents than thosegenerated by existing methods.

Mammalian development follows a carefully orchestrated unfolding of cellfate transitions leading to a complex set of highly specialized celltypes. These cell fate transitions involve the silencing of previouslyactive molecular programs along with the activation of new ones. MiRNAsare small non-coding RNAs that are well suited to suppress previouslyactive programs and, thereby, provide robustness to cell fate decisions(Babiarz, J. E. & Blelloch, R. Small RNAs—their biogenesis, regulationand function in embryonic stem cells. StemBook, ed. The Stem CellResearch Community, StemBook, doi/10.3824/stembook. 1.47.1. (2009).Hornstein, E. & Shomron, N. Canalization of development by microRNAs.Nat. Genet 38, S20-24 (2006). MiRNAs identify their targets via basepairing of nucleotides 2-8 of the miRNA (the seed sequence) withcomplementary sequences within the target mRNA's open reading frame(ORF) and 3′ untranslated region (UTR) (The Stem Cell ResearchCommunity, StemBook, doi/10.3824/stembook. 1.47.1. (2009)). Thistargeting is carried out in coordination with the RNA-induced silencingcomplex (RISC) and often results in both destabilization andtranslational inhibition of the targets. While inhibition of any onetarget is usually only partial, each miRNA binds and suppresses hundredsof mRNA targets, resulting in large overall changes in the molecularconstitution of cells.

BACKGROUND OF THE INVENTION

Differentiated cells are capable of being reprogrammed to anembryonic-like state by transfer of nuclear contents into oocytes or byfusion with embryonic stem (ES) cells. Additionally, studies have shownthat mouse embryonic or adult fibroblasts can be induced to becomepluripotent cells by using retroviral vectors to induce expression offour factors: Oct3/4, Sox2, c-Myc and Klf3. These induced cells aretermed induced pluripotent stem cells (iPS). The iPS cells exhibit themorphology and growth properties of ES cells and express ES cell makergenes. Upon injection into nude mice these cells form tumors, severelylimiting their potential use as therapeutic agents. (See, e.g.,Takahashi, K. and Yamanaka, S., Induction of Pluripotent Stem Cells frommouse Embryonic and Adult Fibroblast Cultures by Defined Factors, Cell,126:663-676 (2006)).

MicroRNAs (miRNAs) are single-stranded RNA molecules, typically between21 and 23 nucleotides in length. They are endogenously occurring,untranslated RNA molecules involved in regulation of gene expression.miRNAs are also important regulators of development and differentiation.(See, e.g., Suh, et al., Human embryonic stem cells express a unique setof miicroRNAs, Developmental biology, 270:488-498 (2004). miRNAsfunction to regulate gene expression through targeting mRNAs forcleavage or translation repression. (See, e.g., Bartel, D. P.,MicroRNAs: Genomics, Biogenesis, Mechanism, and Function Cell116:281-297 (2004).) At present, nearly all of the identified miRNAs areconserved in closely related animals, such as humans and mouse. (See,e.g., Lagos-Quintana et al., New microRNAs from mouse and human RNA9:175-179 (2003); Lim et al., Vertebrate microRNA genes Science 299:1540(2003)).

One microRNA cluster, designated the miR-290 cluster, constitutes over70% of the entire miRNA population in mouse ES cells (Marson, A. et al.Connecting microRNA genes to the core transcriptional regulatorycircuitry of embryonic stem cells Cell 134:521-533 (2008)). Expressionof the miR-290 cluster is rapidly down-regulated upon ES celldifferentiation (See, e.g., Houbaviy, H. B., Murray, M. F. & Sharp, P.A. Embryonic stem cell-specific MicroRNAs Dev Cell 5:351-358 (2003)). Asubset of the miR-290 cluster, called the embryonic stem cell cycle(ESCC) regulating miRNAs, enhances the unique stem cell cell cycle andincludes miR-291-3p, miR-294, and miR-295, as well as the humanhomologues hsa-mir-302a, hsa-miR-302b, hsa-miR-302c, hsa-miR-302d,hsa-miR-371-5p, hsa-miR-372, hsa-miR-373. (See, e.g., Wang, Y. et al.Embryonic stem cell-specific microRNAs regulate the G1-S transition andpromote rapid proliferation Nat Genet 40:1478-1483 (2008)). This subsetincludes miR-291-3p, miR-294, and miR-295 and their homologues.

Removal of genes required for maturation of all miRNAs has shown thatmiRNAs play essential roles in the proliferation and differentiation ofEmbryonic Stem Cells (ESCs)(Wang, Y. et al., Nat Genet 39:380-5 (2007);Kanellopoulou, C. et al. Genes Dev 19:489-501 (2005); Murchison, E. P.et al., Proc Natl Acad Sci USA 102:12135-40 (2005)). For example, theloss of the RNA binding protein DGCR8, which is required for theproduction of all canonical miRNAs, results in a cell cycle defect andan inability to silence the self-renewal program of ESCs when they areplaced in differentiation-inducing conditions (Wang, Y. et al., NatGenet 39:380-5 (2007). The introduction of individual members of afamily of miRNAs, the ESCC miRNAs, into Dgcr8−/− ESCs can rescue thecell cycle defect (Wang, Y. et al., Nat Genet, 40:1478-1483 (2008)). Wehave discovered that these same miRNAs are able to enhance thede-differentiation of somatic cells to iPS cells and also haveidentified another large family of miRNAs, the let-7 family, whichperforms the opposite role to the ESCC family. When introduced intoDgcr8−/− ESCs, let-7 silences self-renewal by suppressing many of thesame downstream targets that are indirectly activated by the ESCCfamily. Indeed, co-introduction of the ESCC miRNAs inhibits the capacityof let-7 to silence self-renewal, and suppression of the let-7 family insomatic cells promotes de-differentiation.

BRIEF SUMMARY OF THE INVENTION

The present invention is based on findings that introduction ofphysiologically relevant miRNAs can affect somatic cell reprogramming.

In a first aspect, the invention provides that the mouse embryonic stem(ES) cell specific microRNAs (miRNA) miR-291-3p, miR-294, and miR-295enhanced the efficiency of Klf4, Oct4 and Sox2 induced pluripotency. Theinvention further demonstrates that these miRNAs did not further enhancereprogramming in the presence of cMyc. As cMyc binds the promoter ofthese miRNAs, these microRNAs may be downstream effectors of cMycpromoted pluripotency. Unlike exogenous cMyc, these miRNAs induced ahomogeneous population of reprogrammed colonies suggesting overlappingand independent functions of cMyc and the miRNAs. Further, as microRNAscan be introduced without the use of retroviral vectors, they exhibitsignificant potential for use as therapeutic agents.

In this aspect, the present invention is based on a method for inducingpluripotency in an isolated cell comprising introducing aphysiologically relevant miRNA into said cell. In some embodiments themiRNA contains the seed sequence AAGUGCU (SEQ ID NO:15) or AAGUGC. Insome embodiments the miRNA contains the seed sequence AAAGUGC (SEQ IDNO: 16). In some embodiments the miRNA affects cell reprogramming. Insome embodiments the effect on cell reprogramming induces pluripotencyin the cell. In some embodiments the effect on cell reprogramminginduces de-differentiation in the cell. In some embodiments the effecton cell reprogramming induces partial de-differentiation. In someembodiments the effect on cell reprogramming inducestrans-differentiation.

In some embodiments, the miRNA is an embryonic stem cell cycle (ESCC)regulating miRNA. In some embodiments the mi RNA is a member of theembryonic stem cell cycle (ESCC) regulating miR-290 cluster.

In some embodiments of the present invention the miRNA is a human miRNA.In some embodiments the miRNA is a mouse miRNA. In some embodiments themiRNA of the present invention is one of mir-291-3p (SEQ ID NO:1),miR-294 (SEQ ID NO:2) miR-295 (SEQ ID NO:3), miR-302d (SEQ ID NO:4),miR-292-3p (SEQ ID NO:5), hsa-mir-302a (SEQ ID NO:8), hsa-miR-302b (SEQID NO:9), hsa-miR-302c (SEQ ID NO:10), hsa-miR-302d (SEQ ID NO:11),hsa-miR371-5 (SEQ ID NO:12), hsa-miR-372 (SEQ ID NO:13), hsa-miR-373(SEQ ID NO:14), sa-miR-17 (SEQ ID NO:17), hsa-miR-20a (SEQ ID NO:18),hsa-miR-20b (SEQ ID NO: 19), hsa-miR-93 (SEQ ID NO:20), hsa-mir-106a(SEQ ID NO:21), or hsa-mir-106b (SEQ ID NO:22).

In some embodiments of the methods of the present invention the cell isa human cell. In some embodiments of the present invention the cell canbe any of the cells typically utilized in generating cells that harborrecombinant nucleic acid constructs. In some embodiments the cells canbe cultured cells or cell lines such as but not limited to COS, CHO,HeLa, 293T or mouse embryonic fibroblasts (MEFs). In some embodimentsthe cell types utilized for the methods of the present invention caninclude naturally occurring cells isolated from tissue samples includingbut not limited to blood, bone, brain, kidney, muscle, spinal cord,nerve, endocrine system, uterine, ear, foreskin, liver, intestine,bladder or skin. In some embodiments the cells can include neural cells,lymphocytes, epidermal cells, islet cells, intestinal cells orfibroblasts. In some embodiments the cells of the present invention canbe autologous or heterologous cells. In some embodiments the cellsuseful for the methods of the present invention can include animalcells. In some embodiments the cells are mammalian. In some embodimentsthe cells are diseased human cells, such as cancer cells. In someembodiments the cancer cells can include but are not limited to breast,prostate, liver, bladder, brain, blood or bone cancer cells.

In some embodiments of the present invention the miRNA is 80% or moreidentical to one of miR-291-3p (SEQ ID NO:1), miR-294 (SEQ ID NO:2),miR-295 (SEQ ID NO:3), miR-302d (SEQ ID NO:4), miR-292-3p (SEQ ID NO:5),hsa-mir-302a (SEQ ID NO:8), hsa-miR-302b (SEQ ID NO:9), hsa-miR-302c(SEQ ID NO:10), hsa-miR-302d (SEQ ID NO:11), hsa-miR371-5 (SEQ IDNO:12), hsa-miR-372 (SEQ ID NO:13), hsa-miR-373 (SEQ ID NO:14),hsa-miR-17 (SEQ ID NO:17), hsa-miR-20a (SEQ ID NO:18), hsa-miR-20b (SEQID NO:19), hsa-miR-93 (SEQ ID NO:20), hsa-mir-106a (SEQ ID NO:21), orhsa-mir-106b (SEQ ID NO:22). In some embodiments of the presentinvention the miRNA contains the seed sequence AAGUGCU (SEQ ID NO:15).In some embodiments of the present invention the miRNA contains the seedsequence AAAGUGC (SEQ ID NO:16). In some embodiments of the presentinvention the miRNA is 80% or more identical to one of miR-291-3p (SEQID NO:1), miR-294 (SEQ ID NO:2), miR-295 (SEQ ID NO:3), miR-302d (SEQ IDNO:4), hsa-mir-302a (SEQ ID NO:8), hsa-miR-302b (SEQ ID NO:9),hsa-miR-302c (SEQ ID NO:10), hsa-miR-302d (SEQ ID NO: 11), hsa-miR371-5(SEQ ID NO: 12), hsa-miR-372 (SEQ ID NO:13), hsa-miR-373 (SEQ ID NO:14),hsa-miR-17 (SEQ ID NO:17), hsa-miR-20a (SEQ ID NO:18), hsa-miR-20b (SEQID NO:19), hsa-miR-93 (SEQ ID NO:20), hsa-mir-106a (SEQ ID NO:21), orhsa-mir-106b (SEQ ID NO:22), and contains the seed sequence AAGUGCU (SEQID NO: 15). In some embodiments of the present invention the miRNA is80% or more identical to one of miR-291-3p (SEQ ID NO: 1), miR-294 (SEQID NO:2), miR-295 (SEQ ID NO:3), miR-302d (SEQ ID NO:4), hsa-mir-302a(SEQ ID NO:8), hsa-miR-302b (SEQ ID NO:9), hsa-miR-302c (SEQ ID NO:10),hsa-miR-302d (SEQ ID NO: 11), hsa-miR371-5 (SEQ ID NO:12), hsa-miR-372(SEQ ID NO:13), hsa-miR-373 (SEQ ID NO:14), hsa-miR-17 (SEQ ID NO:17),hsa-miR-20a (SEQ ID NO:18), hsa-miR-20b (SEQ ID NO:19), hsa-miR-93 (SEQID NO:20), hsa-mir-106a (SEQ ID NO:21), or hsa-mir-106b (SEQ ID NO:22),and contains the seed sequence AAAGUGC (SEQ ID NO: 16).

In some embodiments of the present invention the miRNA is a member ofmiR-290 cluster. In some embodiments the miRNA is one of miR-291-3p (SEQID NO:1), miR-294 (SEQ ID NO:2) miR-295 (SEQ ID NO:3), miR-302d (SEQ IDNO:4), miR-292-3p (SEQ ID NO:5), hsa-mir-302a (SEQ ID NO:8),hsa-miR-302b (SEQ ID NO:9), hsa-miR-302c (SEQ ID NO: 10), hsa-miR-302d(SEQ ID NO: 11), hsa-miR371-5 (SEQ ID NO:12), hsa-miR-372 (SEQ IDNO:13), or hsa-miR-373 (SEQ ID NO:14).

In some embodiments of the present invention the effect on cellreprogramming is to enhance reprogramming. In some embodiments methodsof the present invention include inducing pluripotency in a cell, suchthat the cell becomes capable of dividing and differentiating into anycell type other than embryonic cells. In some embodiments cellularreprogramming can include inducing pluripotency in or de-differentiationof the cell. In some embodiments altering cell reprogramming can referto enhancing the level of pluripotency or de-differentiation that hasbeen induced by an agent other than a microRNA. In some embodiments, thepluripotent or multipotent cells, also called stem cells, have theability to divide (self-replicate or self-renew) or differentiate intomultiple different phenotypic lineages for indefinite periods. In someembodiments, the cells of the present invention when under specificconditions, or in the presence of optimal regulatory signals, can becomepluripotent and differentiate themselves into many different cell typesthat make up the organism. In some embodiments the pluripotent ormultipotent cells of the present invention possess the ability todifferentiate into mature cells that have characteristic attributes andspecialized functions, such as hair follicle cells, blood cells, heartcells, eye cells, skin cells, pancreatic cells, or nerve cells. In someembodiments of the present invention cell reprogramming can furtherinclude partial dc-differentiation to a closely related cell or celltype. In some embodiments cell reprogramming can also includetrans-differentiation, wherein a cell of the present invention convertsfrom one differentiated cell type into another differentiated cell type.In some embodiments of the above the cell expresses, or was transducedwith retroviruses expressing Oct4, Sox2, and Klf4 (OSK).

In some embodiments of the present invention the enhancement of cellreprogramming constitutes enhancement of pluripotency. In someembodiments the enhancement of cell reprogramming constitutesenhancement of de-differentiation.

In some embodiments the isolated cell of the present invention containsone miRNA. In some embodiments the isolated cell of the presentinvention contains more than one miRNA. In some embodiments the isolatedcell of the present invention contain one miRNA, wherein the miRNAcontains the seed sequence AAGUGCU (SEQ ID NO: 15) or AAGUGC. In someembodiments the isolated cell of the present invention contains onemiRNA, wherein the miRNA contains the seed sequence AAAGUGC (SEQ ID NO:16). In some embodiments the isolated cell of the present inventioncontains more than one miRNA, wherein the miRNA contains the seedsequence AAGUGCU (SEQ ID NO:15) or AAGUGC. In some embodiments theisolated cell of the present invention contains more than one miRNA,wherein the miRNA contains the seed sequence AAAGUGC (SEQ ID NO: 16). Insome embodiments of the present invention the miRNA containing cells arein cell culture. In some embodiments of the present invention the miRNAcontaining cells are in cell culture and contain one or more of themiRNAs.

In embodiments of methods of the present invention the methods includetreating an individual with a degenerative disease. In some embodimentsof the methods of the present invention comprise treating an individualby administering to an individual a cell containing an miRNA of thepresent invention. In some embodiments the miRNA contains the seedsequence AAGUGCU (SEQ ID NO: 15) or AAGUGC. In some embodiments themiRNA contains the seed sequence AAAGUGC (SEQ ID NO: 16). In someembodiments the miRNA affects cell reprogramming. In some embodimentsthe effect on cell reprogramming induces pluripotency in the cell. Insome embodiments the effect on cell reprogramming inducesde-differentiation in the cell. In some embodiments the effect on cellreprogramming induces partial de-differentiation. In some embodimentsthe effect on cell reprogramming induces trans-differentiation. In someembodiments of the present invention the miRNA is 80% or more identicalto one of miR-291-3p (SEQ ID NO:1), miR-294 (SEQ ID NO:2), miR-295 (SEQID NO:3), miR-302d (SEQ ID NO:4), miR-292-3p (SEQ ID NO:5), hsa-mir-302a(SEQ ID NO:8), hsa-miR-302b (SEQ ID NO:9), hsa-miR-302c (SEQ ID NO:10),hsa-miR-302d (SEQ ID NO:11), hsa-miR371-5 (SEQ ID NO:12), hsa-miR-372(SEQ ID NO:13), hsa-miR-373 (SEQ ID NO: 14), hsa-miR-17 (SEQ ID NO:17),hsa-miR-20a (SEQ ID NO:18), hsa-miR-20b (SEQ ID NO:19), hsa-miR-93 (SEQID NO:20), hsa-mir-106a (SEQ ID NO:21), or hsa-mir-106b (SEQ ID NO:22).In some embodiments of the present invention the miRNA contains the seedsequence AAGUGCU (SEQ ID NO:15) or AAGUGC. In some embodiments of thepresent invention the miRNA contains the seed sequence AAAGUGC (SEQ IDNO:16). In some embodiments of the present invention the miRNA is 80% ormore identical to one of miR-291-3p (SEQ ID NO: 1), miR-294 (SEQ IDNO:2), miR-295 (SEQ ID NO:3), miR-302d (SEQ ID NO:4), miR-292-3p (SEQ IDNO:5), hsa-mir-302a (SEQ ID NO:8), hsa-miR-302b (SEQ ID NO:9),hsa-miR-302c (SEQ ID NO:10), hsa-miR-302d (SEQ ID NO: 11), hsa-miR371-5(SEQ ID NO:12), hsa-miR-372 (SEQ ID NO: 13), hsa-miR-373 (SEQ ID NO:14),hsa-miR-17 (SEQ ID NO:17), hsa-miR-20a (SEQ ID NO:18), hsa-miR-20b (SEQID NO:19), hsa-miR-93 (SEQ ID NO:20), hsa-mir-106a (SEQ ID NO:21), orhsa-mir-106b (SEQ ID NO:22), and contains the seed sequence AAGUGCU (SEQID NO:15) or AAGUGC. In some embodiments of the present invention themiRNA is 80% or more identical to one of miR-291-3p (SEQ ID NO:1),miR-294 (SEQ ID NO:2), miR-295 (SEQ ID NO:3), miR-302d (SEQ ID NO:4),miR-292-3p (SEQ ID NO:5), hsa-mir-302a (SEQ ID NO:8), hsa-miR-302b (SEQID NO:9), hsa-miR-302c (SEQ ID NO:10), hsa-miR-302d (SEQ ID NO: 11),hsa-miR371-5 (SEQ ID NO:12), hsa-miR-372 (SEQ ID NO: 13), hsa-miR-373(SEQ ID NO:14), hsa-miR-17 (SEQ ID NO:17), hsa-miR-20a (SEQ ID NO:18),hsa-miR-20b (SEQ ID NO:19), hsa-miR-93 (SEQ ID NO:20), hsa-mir-106a (SEQID NO:21), or hsa-mir-106b (SEQ ID NO:22), and contains the seedsequence AAAGUGC (SEQ ID NO:16). The treatments may be ex vivo or invivo. In some embodiments of any of the above, the cells are autologous.For instance, in some embodiments, the cells may be obtained from theintended recipient having the disease, treated with the miRNA in orderto induce pluripotency or reprogram the cells, and returned to theintended recipient.

In some embodiments of the methods of the present invention, the methodsinclude treating and individual with a degenerative disease. In someembodiments the degenerative disease includes but is not limited toParkinson's Disease, Alzheimer's Disease, skin grafts, MuscularDystrophy, Amyotrophic Lateral Sclerosis (ALS) (e.g., Lou Gehrig'sDisease), Multiple system atrophy, Niemann Pick disease,Atherosclerosis, Progressive supranuclear palsy, cancer, metabolicdiseases (including for example but not limited to Tay-Sachs Disease),Diabetes, Heart Disease, Inflammatory Bowel Disease (IBD), Norricdisease, Prostatitis, Osteoarthritis, Osteoporosis, RheumatoidArthritis, Sickle Cell Anemia, heart disease, spinal and nerve relateddiseases and disorders (including spinal and nerve related injuries),cancers (including for example but not limited to leukemias andlymphomas), other injuries induced by trauma, as well as regeneration oftissue post-resectioning.

In some embodiments of any of the above, the miRNA is an miRNA set forthin Example 3 or 4.

In a second aspect, the invention provides a method of stabilizing asomatic cell in a differentiated state or of reducing the proliferationrate of a cell by administering a Let-7 miRNA or a miRNA having the seedsequence of Let-7 (e.g., mir-98, (ugagguaguaaguuguauuguu)) to the celland/or by administering an inhibitor of an miRNA according to SEQ IDNOs: 1 to 22 or an inhibitor of an miRNA having the seed sequence of SEQID NOs: 15 or 16 or AAGUGC, or administering an miRNA set forth inExamples 5 or 6. In some embodiments, one, two, three or more of thesemiRNA and/or their inhibitors is administered to the cell. In someembodiments, the Let-7 miRNA is selected from the group consisting ofLet-7a, Let-7b, Let-7c, Let-7d, Let-7e, Let-7f, and Let-7g. In someembodiments, the invention provides a method of treating a subjecthaving a disease mediated by proliferating cells (e.g., cancer, andautoimmune disease) by administering to the subject a therapeuticallyeffective amount of a Let-7 miRNA or a miRNA set forth in Example 5and/or administering a therapeutically effective amount of an inhibitorof an miRNA according to SEQ ID NOs: 1 to 22 or an inhibitor of an miRNAhaving the seed sequence of SEQ ID NOs: 15 or 16 or of AAGUGC.

In another embodiment of this second aspect, the invention provides amethod of inducing pluripotency in a somatic cell by administering aninhibitor of a Let-7 miRNA or of an miRNA of Example 5 to the celland/or by administering an miRNA according to SEQ ID NOs: 1 to 22 or aninhibitor of an miRNA having the seed sequence of SEQ ID NOs: 15 and 16or of AAGUGC. In some embodiments of the methods of the presentinvention, the methods include treating an individual with adegenerative disease by administering a therapeutically effective amountof an inhibitor of a Let-7 miRNA or a miRNA having the seed sequence ofLet-7 (e.g., mir-98, (ugagguaguaaguuguauuguu)) or an inhibitor of anmiRNA of Example 5 or 6 to the cell and/or by administering atherapeutically effective amount of an miRNA according to SEQ ID NOs: 1to 22 or of Examples 3 or 4 or of an miRNA having the seed sequence ofSEQ ID NOs: 15 and 16 or of AAGUGC. In some embodiments, one, two, threeor more of these miRNA or their inhibitors is administered. In someembodiments, the degenerative disease includes but is not limited toParkinson's Disease, Alzheimer's Disease, skin grafts, MuscularDystrophy, Amyotrophic Lateral Sclerosis (ALS) (e.g., Lou Gehrig'sDisease), Multiple system atrophy, Niemann Pick disease,Atherosclerosis, Progressive supranuclear palsy, cancer, metabolicdiseases (including for example but not limited to Tay-Sachs Disease),Diabetes, Heart Disease, Inflammatory Bowel Disease (IBD), Norriedisease, Prostatitis, Osteoarthritis, Osteoporosis, RheumatoidArthritis, Sickle Cell Anemia, heart disease, spinal and nerve relateddiseases and disorders (including spinal and nerve related injuries),cancers (including for example but not limited to leukemias andlymphomas), other injuries induced by trauma, as well as regeneration oftissue post-resectioning.

In another aspect the invention provides a method of supporting,promoting, or stabilizing the differentiation of, or preventing theproliferation of a cell, comprising administering a micronucleic acid orinhibitor (e.g. a member of the let-7 family or a miRNA having the seedsequence of Let-7 (e.g., mir-98, (ugagguaguaaguuguauuguu)); an miRNA ofExample 5 an miRNA of FIG. 20, an ESCC miRNA inhibitor, or an inhibitorof any miRNA of Example 4) to the cell. In some embodiments, the cell isan induced pluripotent cell, an embryonic stem cell or is derived froman iPS, or ES cell. In some embodiments of any of the above, the nucleicacid is administered to cells in culture or is administered systemicallyto a patient, with the intention of targeting the cells. In someembodiments, one, two, three or more of these miRNA and/or theirinhibitors is administered. In some embodiments the cells are furtherintroduced into a mammalian host (with a greatly reduced likelihood ofany cells therein would be capable of causing a teratoma in the host.The cells may be heterologous or autologous with regard to the host orpatient. The cells may be introduced to treat a degenerative conditionin the host or restore a cellular function or cell type deficient in thehost. In some embodiments, the invention provides cells which have beenobtained by administering the micronucleic acid or inhibitor to thecell.

In any of the above embodiments and aspects, the miRNA can be introducedas a pre- or primary miRNA. In some embodiments, an miRNA or inhibitorthereof from Examples 3 to 6, SEQ ID NOs: 1 to 14, or having the seedsequence of SEQ ID NOs 15 and 16 or of AAGUGC or let-7 is used toreprogram a cell. In some embodiments, one two, three or more membersselected from the miRNAs and inhibitors are used together orsequentially. In some embodiments, the members each act to either topromote de-differentiation of a cell. In other embodiments, the memberseach act to support, promote, or stabilize the differentiation of, orprevent the proliferation of a cell. In still other embodiments, somemembers are used first to de-differentiate a cell and other members arelater used to re-differentiate, support, promote, or stabilize thedifferentiation of, or prevent the proliferation of a cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The ESCC miRNAs promote three-factor, but not four-factorinduced pluripotency. (a) Fold increase of day 10 Oct-GFP+ colonies withretroviruses expressing Oct4, Sox2, and Klf4 (OSK) together with 16 nMmiRNA mimic relative to transfection reagent only (Mock). N=3. Raw datain Table 1. (b) Sequence of miR-290 cluster, miR-302d, and miR-294 seedsequence mutant. Bold indicates seed sequence. Capitals indicate pointmutations. Grey box highlights ESCC seed-sequence. (c) Fold increase inday 10 Oct4-GFP+ colonies with addition of mimic to OSK in the presence(light grey) or absence (dark grey) of cMyc retrovirus. Bars representthe number of GFP+ colonies after mimic transfection divided by thenumber of GFP+ colonies after mock transfection. N=6, 26, 2, 5, & 3 leftto right. Asterisk indicates p-value≦0.0001. Raw data for bars 1&2 inTable 2. (d) Percent day 10 Oct4-GFP colonies for OSK plus 1.6, 16 and160 nM transfected miR-294 mimic or 160 nM miR-1 relative to OSKM. (e)Quantitative RT-PCR for endogenous pluripotency markers in control(V6.5) ES cells, MEFs, and miR-294-iPS lines. N=3, 3, & 5. RPL7 was usedas input control. Data was normalized to ES expression. (f) QuantitativeRT-PCR for exogenous Oct4, Sox2, and Klf4 in MEFs 6 days after viralinfection, control (V6.5) ES cells, and MEFs (each N=3) and 5 individualmiR-294-iPS lines. Horizontal black bars indicate Ct>40. RPL7 was usedas input control. Data was normalized to MEF expression 6 days afterviral infection. (g) X-gal staining demonstrates miR-294-iPS chimericcontribution to ectoderm (neural tissue, N), endoderm (lung, L), andmesoderm (cartilage, C). (h) GFP expression in genital ridges of E12.5chimera demonstrates Oct4-GFP miR-294-iPS contribution to germline. Allerror bars indicate standard deviation.

FIG. 2. Characterization of the relationship between Myc and miR-294.(a) cMyc (blue) and nMyc (yellow) bind the miR-290 cluster promoter.ChlP-seq data reads (Chen, X. et al., Integration of external signalingpathways with the core transcriptional network in embryonic stem cells.Cell 133, 1106-1117 (2008)) were aligned to the mm9 assembly of thegenome and peaks were generated with Findpeaks (Fejes, A. P. et al.FindPeaks 3.1: a tool for identifying areas of enrichment from massivelyparallel short-read sequencing technology. Bioinformatics 24, 1729-1730(2008)). Vertical hash marks denote the positions of the miR-290 clustermiRNAs. (b) Quantitative RT-PCR for total mature miR-294 expression incontrol (V6.5) ES cells, MEFs, and MEFs infected with viruses expressingSox2 (S), Oct4 (0), Klf4 (K) or cMyc (M). RNA was collected on days 2and 6. N=3. Horizontal black bars indicate Ct>40. Sno202 was used asinput control. Data was normalized to ES cells. (c) H3K4me3 (green) andH3K27me3 (red) surrounding the miR-290 cluster in MEFs. Chip-seq data(Mikkelsen, T. S. et al. Genome-wide maps of chromatin state inpluripotent and lineage-committed cells. Nature 448, 553-560 (2007))were analyzed as described in a. (d) Quantitative RT-PCR for totalmature miR-294 expression in control (V6.5) ES cells (E), MEFs (M), MEFsinfected with either Oct4, Sox2, Klf4 (OSK); Oct4, Sox2, Klf4, and cMyc(OSKM); or OSK+miR-294, and established iPS lines resulting from theseconditions (iPS). RNA was collected on days 2, 6, and 10 ofreprogramming. Three independent experiments are shown. Horizontal blackbars indicate Ct>40. Sno202 was used as input control. Data wasnormalized to ES cells. (e) Total cell number during reprogramming.Cells were counted on day 7 after infection with OSKM or OSK+/−miRNAmimic. Concentrations of miR-294 mimic: 1.6, 16 and 160 nM.Concentration of miR-1 mimic: 160 nM. (f) GFP negative colonies inpresence of cMyc. Oct4-GFP+, ES-like colonies (black arrow) andGFP-negative, non-ES-like colonies (white arrow). (g) Quantification ofnumber of day 10 GFP-negative colonies after infection with OSKM orOSK+/−miR-294 mimic. All error bars indicate standard deviation of N=3.

FIG. 3. Characterization and application of miR-290 cluster mimics. (a)miR-290 cluster expression levels in mimic-transfected MEFs. MiRNA mimic(16 nM) was transfected into MEFs, and RNA was collected on days 1, 2,3, and 6. Relative miRNA levels were compared via RT-qPCR to control(v6.5) ES cells (black horizontal bar). Mimic levels were found to bewell above ES expression levels one day after transfection, but close tophysiological levels between days 2 and 3. (b) Reprogramming assaytimeline. In reprogramming assays, MEFs were transfected on days 0 and 6in order to retain ES-like levels of the mimics.

FIG. 4. Generation of GFP+ colonies with retroviruses expressing Oct4,Sox2, and Klf4 together with either duplex miR-294 mimic (16 nM),hairpin miR-294 mimic (16 nM and 160 nM) or transfection reagent only(mock). Error bars indicate standard deviation of N=3.

FIG. 5. Effect of combining ESCC miRNAs on reprogramming. Generation ofGFP+ colonies with retroviruses expressing Oct4, Sox2, and Klf4 (OSK)together with either miR-294 (16 nM or 48 nM) or a mixture ofmiR-291-3p, miR-294 and miR-295 (5.3 nM each or 16 nM each) ortransfection reagent only (mock).

FIG. 6. FACS analysis of GFP+ cells from MEFs infected with Oct4, Sox2,and Klf4 (OSK) and transfected with miRNA mimics. (a) Graphic display ofFACS analysis for GFP+ sorted cells on day 12. Wedge indicatesincreasing concentrations (1.6, 16 and 160 nM) of mimic. N=3. Error barsindicate standard deviation. (b) FACS plots of representative samples.

FIG. 7. Kinetics of reprogramming and effects of miR-294 on othercombinations of transcription factors. (a) Generation of GFP+ colonieswith retroviral expression of Oct4, Sox2, and Klf4 alone (OSK), with theaddition of cMyc (OSKM), or with transfection of 16 nM miR-294 mimic(OSK+miR-294). GFP+ colonies were counted on days 5-10. GFP+microcolonies were reliably first seen in OSKM and OSK+miR-294 by day 7with identifiable GFP+ES-like colonies seen by day 9. GFP+ microcoloniesand ES-like colonies were generally seen in OSK on days 8 and 10,respectively. Error bars represent standard deviation for N=3. (b)Generation of GFP+ colonies with combinations of retroviruses expressingOct4, Sox2, Klf4 or cMyc with and without transfection of miR-294 (16nM). Lane 1 depicts a single experiment representative of many (Table2). Lanes 2, 3 and 4 depict N=1, 1, and 3, respectively.

FIG. 8. Verification of miR-294-iPS pluripotency. (a) Brightfield andimmunofluorescent photographs of miR-294-iPS colonies. Images arerepresentative of six independent iPS lines. Staining controls includeES (V6.5) cells, cMyc-iPS colonies, miR-294-iPS colonies with secondaryantibody only, and MEFs (b) Representative karyotype of iPS cellsinduced with Oct4, Sox2, Klf4 and miR-294 mimic. (c) An E15 chimeraderived from blastocyst injection of miR-294-iPS cells carrying aubiquitously expressed—galactosidase reporter.

FIG. 9. Teratoma generation from V6.5, miR-294-iPS, and cMyc-iPS celllines. (A) H&E staining of miR-294-iPS derived teratomas. Images depict,left to right, bone, neural tissue, keratinizing squamous epithelialtissue, and glandular tissue. (B) Left and Middle, images ofrepresentative control ES (V6.5) and miR-294-iPS noninvasive teratomas.Right, image of representative cMyc-iPS invasive teratoma. (C) Number ofinvasive and noninvasive tumors with different cell lines injected.Columns display from left to right, independent cell lines, number ofmice injected, number of total teratomas isolated, number of totalteratomas found to be invasive, and the number of days after injectionteratomas were harvested. Percent teratoma refers to the percentage ofcell lines that formed teratomas. Percent invasive refers to thepercentage of teratomas found to be invasive, defined as migrationthrough the underlying body wall.

FIG. 10. The let-7 and ESCC miRNA families have opposing roles inregulating ESC self-renewal. (a) Transfected miRNAs with the seedsequence highlighted. (b) Pou5fl/Oct4 immunofluorescence staining aftertransfection of let-7c, miR-294 and combinations of let-7c with miR-294,mutant-miR-294, miR-291a-5p, or miR-130b in Dgcr8−/− (i) and wild-type(ii) ESCs. Representative images, n=3. (c) qRT-PCR for Pou5fl/Oct4,Sox2, and Nanog normalized to beta-actin after miRNA introduction as inb. n=3-8. * indicates p<0.02. (d) Colony reforming assays after miRNAintroduction as in b and c. n=3. * indicates p<0.05. All p-valuesgenerated by Bonferroni corrected t-test of comparisons to let-7ctreated. Error bars represent standard deviation.

FIG. 11. The let-7 and ESCC miRNAs suppress hundreds of transcripts bybinding their ORF and/or 3'UTR. (a) Microarray analysis followingintroduction of let-7c alone. Upregulated transcripts are shown in darkgrey, downregulated transcripts in black (FDR<0.05). (b) Analysis ofseed matches in the promoter, 5'UTR, ORF, and 3'UTR oflet-7c-downregulated and upregulated transcripts. Presented are the meannumber of seeds matches per kb of sequence for the listed groups ofaltered genes described in a. P-values calculated by the Wilcoxon RankSum Test and Bonferroni corrected are shown for p<0.01 (c) Microarrayanalysis following introduction of miR-294 alone. Color labeling, as ina. (d) Seed analysis as in b for miR-294 up and downregulatedtranscripts.

FIG. 12. Enrichment/depletion of transcription factor bound genes amongmiRNA-regulated transcripts. (a) A schematic of hypothetical miRNAregulation of a transcription factor or its targets. Correspondingexpected enrichment/depletion of the transcription factor ChIP targetsin miRNA-induced upregulated or downregulated transcript sets aredisplayed in a heat map. A key of color coding representing relativeenrichment is given in b. (b) A heat map showing enrichment of the ChIPtargets among the different sets of miRNA-regulated transcripts on thehorizontal axis. Vertical axis represents the different ChIP data setswith first author and factor that was immunoprecipitated.

FIG. 13. Let-7c and miR-294 regulate Lin28, Sa114, cMyc, and nMyc. (a)qRT-PCR for Lin28, Sa114, nMyc, and cMyc 12 hours after transfectionwith let-7c, miR-294, or a combination of the two. n=3. (b)Representative Western blot analysis 48 hours after transfection withmiRNAs. (c) Luciferase analysis of Sa114 and nMyc 3'UTRs. Seed matchesfor let-7c in the 3'UTRs along with different mutant constructs arediagrammatically represented in the left panel. Luciferase results afterco-transfection with let-7c mimic releative to mock transfected areshown in the right panel. All data are represented as mean+/− standarddeviation. * indicates p<0.05 by Bonferroni corrected t-test.

FIG. 14. Inhibition of let-7 miRNAs promotes reprogramming to inducedpluripotency (a) Fold increase of Oct4::GFP positive colonies inreprogramming with transduction of 3TFs (Pou5fl/Oct4, Sox2, and Klf4) or4TFs (+cMyc) after mock, let-7 inhibitor, or control inhibitortransfection. P-values are indicated for p<0.01 calculated by Bonferronicorrected t-test. n=10 for mock and let-7 inhibitor samples and n=6 forcontrol inhibitor samples (b) A model of the antagonism between themiR-294 and let-7c in the stabilization of the self-renewing anddifferentiated states. Bold and enlarged genes and arrows are active inthe indicated state. Mechanisms of ESCC upregulation of Lin28 and cMycare unknown and represented by a question mark.

FIG. 15. The let-7 family of miRNAs function to suppress self-renewal inDgcr8−/− ESCs. qRT-PCR for Oct4, Sox2, and Nanog normalized tobeta-actin after transfection with different let-7 family members eitheralone or in combination with miR-294. n=3, error bars represent standarddeviation

FIG. 16. miR-290 cluster ESCC family members function to suppress let-7cinduced silencing of ESC self-renewal in Dgcr8−/− ESCs. qRT-PCR forOct4, Sox2, and Nanog normalized to beta-actin after transfection withdifferent ESCC family members either alone or in combination withlet-7c. n=3, error bars represent standard deviation.

FIG. 17. The let-7 inhibitor reduces levels of the majority of maturelet-7 family miRNAs. polyA miRNA qPCR was performed in mock, controlinhibitor, and let-7 inhibitor treated MEFs. Let-7 family miRNAs but notother highly expressed miRNAs (miR-19b, miR-26a, and miR-34c) havereduced levels in the presence of the let-7 inhibitor.

FIG. 18. miR-372 enhances 4 factor reprogramming of human foreskinfibroblasts Human foreskin fibroblasts were infected with retrovirusesexpressing Oct4, Sox2, Klf4, c-Myc and a fluorescent protein, Venus.MicroRNA 372 was transfected on days 3 and 10 post infection.Introduction of microRNA 372 resulted in an increase in the number ofhuman ES cell-like colonies. Silencing of exogenous factors, which is anindication of complete reprogramming was assessed by the silencing ofVenus.

FIG. 19. miR-372 and 302b enhance 3 factor reprogramming of humanforeskin fibroblasts. Human foreskin fibroblasts were infected withretroviruses expressing Oct4, Sox2, Klf4 and a fluorescent protein,Venus. MicroRNA 372, 302b or 372 and 302b were transfected on days 3 and10 post infection. The number of human ES cell-like colonies werecounted on days 17 and 20 post infection. Silencing of exogenousfactors, which is an indication of complete reprogramming was assessedby the silencing of Venus.

FIG. 20. Results of a screen assay for loss of ESC self-renewal inDgcr8−/− ES cells after addition of individual miRNA mimics. Impact ofmiRNAs on ESC self-renewal was assayed by staining for alkalinephosphatase followed by blind scoring of amount of staining from 1-6. Ascore of 1 indicates the least amount of alkaline phosphatase stainingand therefore the most silencing of ESC self-renewal. A score of 6indicates high alkaline phosphatase staining and no silencing of ESCself-renewal. The screen was repeated in triplicated. Shown here are themean of the triplicate+−standard deviation. Only miRNAs with a score ator below 3 are included in this figure. MiRNAs shown here are capable ofsilencing ESC self-renewal in Dgcr8−/− ESCs.

FIG. 21. Results of an experiment introducing select screen positivemiRNAs (from FIG. 20) into Dgcr8−/− ESCs either alone or in combinationwith the ESCC miRNA miR-294. The data are the relative mRNA expressionvalues of oct3/4, sox2, and nanog as measured by qRTPCR 3 days afterintroduction of the miRNA mimics. These data indicate that the screenpositive miRNAs silence ESC self-renewal but only in the absence of ESCCmiRNAs. The findings support silencing ESC self-renewal in ES or iPScells by inhibition of ESCCs in combination with introduction of screenpositive miRNAs.

FIG. 22. Results of an experiment looking for enriched motifs locatedwithin the 3p UTRs of transcripts which were found to be down-regulatedin Dgcr8−/− ESCs 12 hours after introduction of miR-294 miRNA mimic.These motifs were identified as 4 fold or greater enriched in the 3pUTRs of downregulated transcripts compared to all other transcripts onthe microarrays. Motifs were measured for their similarity to oneanother and motifs with 2 or fewer mismatches were clustered together.This particular cluster represents the motifs corresponding to the miRNAseed sequence. The consensus of these motifs is shown in this figure.These data support the idea that the minimal seed sequence of AAGUGC isrequired for ESCC miRNA function.

DETAILED DESCRIPTION

The breadth and importance of miRNA directed gene regulation is becomingmore apparent as more miRNAs and their regulatory targets are identifiedand described. Recently discovered functions include control of cellproliferation, cell death, fat metabolism in nematodes, modulation ofhematopoietic lineage and control of leaf and flower development inplants (see, e.g., Bartel, D. P., MicroRNAs: Genomics, Biogenesis,Mechanism, and Function Cell 116:281-297 (2004)).

The methods of the present invention focus on the induction ormodulation of pluripotency by miRNAs. In some embodiments the miRNAs ofthe invention contain a seed sequence (SEQ ID NO: 15) or AAGUGC. In someembodiments, the miRNAs are embryonic stem cell cycle regulating miRNAs.In some embodiments, miRNAs are members of the embryonic stem cell cycleregulating cluster miR-290. In some embodiments the miRNAs are human ormouse miRNAs. In some embodiments, the miRNAs are selected from thegroup including but not limited to miR-291-3p (SEQ ID NO:1), miR-294(SEQ ID NO:2), miR-295 (SEQ ID NO:3), miR-302d (SEQ ID NO:4), miR-292-3p(SEQ ID NO:5), miR-293 (SEQ ID NO:6), miR-294 mut (SEQ ID NO:7),hsa-mir-302a (SEQ ID NO:8), hsa-miR-302b (SEQ ID NO:9), hsa-miR-302c(SEQ ID NO:10), hsa-miR-302d (SEQ ID NO:11), hsa-miR-371-5p (SEQ ID NO:12), hsa-miR-372 (SEQ ID NO: 13), hsa-miR-373 (SEQ ID NO:14), hsa-miR-17(SEQ ID NO:17), hsa-miR-20a (SEQ ID NO:18), hsa-miR-20b (SEQ ID NO:19),hsa-miR-93 (SEQ ID NO:20), hsa-mir-106a (SEQ ID NO:21), or hsa-mir-106b(SEQ ID NO:22).

Accordingly, the invention provides a method of inducing pluripotency inan isolated cell comprising introducing a small nucleic acid into saidcell, wherein the nucleic acid affects cell reprogramming; therebyinducing pluripotency in a cell. The nucleic acid can be a microRNA ormiRNA mimic. For instance, the miRNA can be a member of the embryonicstem cell cycle (ESCC) miRNAs that include the 290 family 302 cluster,17 to 19 cluster, and mir-106. The miRNA can also be a member of the 370family in humans. In some embodiments, the miRNA contains the seedsequence AAGUGCU (SEQ ID NO:15) or AAGUGC or AAAGUGC (SEQ TD NO: 16) orthe sequence AAGUGC (see, FIG. 22). In other embodiments, the miRNA isany of the miRNAs listed in Example 4. In yet other embodiments, thenucleic acid is an inhibitor of a microRNA (e.g., the small nucleic acidis an inhibitor of the miRNA of the let-7 family). In some furtherembodiments, the nucleic acid inhibits an miRNA listed in Example 5 or asilencing miRNA of FIG. 20. In yet other embodiments, the miRNA beinginhibited is a silencing miRNA of FIG. 20. In some embodiments, one,two, three or more of these miRNA and/or their inhibitors isadministered to the cell. In any of the above embodiments, the miRNA andthe cell is human or mouse. For instance, the isolated cell may be ahuman or mouse fibroblast or keratinocyte. In still other embodiments,the miRNA is (i) substantially identical or 80% or more percentidentical in sequence to one of miR-291-3p(SEQ ID NO:1), miR-294 (SEQ IDNO:2), miR-295 (SEQ ID NO:3), miR-302d (SEQ ID NO:4), miR-292-3p (SEQ IDNO:5), hsa-mir-302a (SEQ ID NO:8), hsa-miR-302b (SEQ ID NO:9),hsa-miR-302c (SEQ ID NO:10), hsa-miR-302d (SEQ ID NO: 11), hsa-miR-372(SEQ ID NO:13), hsa-miR-373 (SEQ ID NO:14), hsa-miR-17 (SEQ ID NO:17),hsa-miR-20a (SEQ ID NO:18), hsa-miR-20b (SEQ ID NO:19), hsa-miR-93 (SEQID NO:20), hsa-mir-106a (SEQ ID NO:21), or hsa-mir-106b (SEQ ID NO:22);or a miRNA of Example 4; and (ii) contains the seed sequence AAGUGCU(SEQ ID NO:15) or AAAGUGC (SEQ ID NO:16) or (iii) contains the sequenceAAGUGC. In some additional embodiments, the nucleic acid is an ESCCmicroRNA selected from miR-291-3p (SEQ ID NO:1), miR-294 (SEQ ID NO:2)miR-295 (SEQ ID NO:3), miR-302d (SEQ ID NO:4), miR-292-3p (SEQ ID NO:5),hsa-mir-302a (SEQ ID NO:8), hsa-miR-302b (SEQ ID NO:9), hsa-miR-302c(SEQ ID NO:10), hsa-miR-302d (SEQ ID NO:11), and could also includemembers of the 370 cluster of microRNAs in the human: hsa-miR371-5 (SEQID NO:12), hsa-miR-372 (SEQ ID NO:13), or hsa-miR-373 (SEQ ID NO: 14).In still other embodiments of any of the above, the effect onreprogramming is to enhance reprogramming (e.g., enhancement ofpluripotency or de-differentiation).

In other further embodiments of the above, one or more reprogrammingfactors are also introduced to the cell. These factors include Oct4,Sox2, and Klf4 (OSK), cMyc, Nr5a2, Essrb, cyclinD1, Nanog, Lin28, Sa114,UTF1 or other members of these families. For instance, the contemplatedfactors include inhibitors to p53, p16, p19, p21 or other familymembers. The factors can be introduced virally, via a plasmid, via atransposon, as a protein, as RNA or DNA encoding the factor. The factorsand small molecules (e.g., VPA, TSA, 5′-azac, kenpaullone,TGFβ-inhibitors, Wnts, vitamin C, BIX-01294, BayK8644), as well as thenucleic acids, can be introduced into the cell directly or to the cellmedia.

Accordingly, in still other embodiments the invention provides isolatedcells produced by the above methods. The cell may, for instance, containone, two, three or more miRNAs containing seed sequence AAGUGCU (SEQ IDNO:15) or AAAGUGC (SEQ ID NO:16) or AAGUGC. The invention also providesa method of treating a patient comprising, 1) obtaining and culturingand/or expanding a population of cells from the patient or compatibledonor (e.g., fibroblasts, keratinocytes, somatic cells) 2)de-differentiating the cells with a mixture of reprogramming factorsincluding ESCC miRNAs and/or Let7 inhibitors to generate iPS cells,optionally further culturing and expanding the iPS cell population, 3)differentiating the iPS into cell types required by the patient; and 4)treating the cells with Let-7miRNA and/or ESCC inhibitors to helpmaintain the differentiated phenotype and/or reduce or prevent thepresence of any pluripotent cells; and administering (e.g., injecting orreturning) the cells to the patient. Most commonly used cells arederived from skin biopsies, hair follicles, newborn foreskin, human cordblood, human fetal neural stem cells of the patient or donor. The cellsare preferably returned or instilled into the tissue or site of theirdeficiency. In some embodiments, the individual has a degenerativedisease. In some such embodiments, the degenerative disease is one ormore of Parkinson's Disease, Alzheimer's Disease, skin grafts, MuscularDystrophy, Amyotrophic Lateral Sclerosis (ALS) (e.g., Lou Gehrig'sDisease), Multiple system atrophy, Niemann Pick disease,Atherosclerosis, Progressive supranuclear palsy, cancer, metabolicdiseases (including for example but not limited to Tay-Sachs Disease),Diabetes, Heart Disease, Inflammatory Bowel Disease (IBD), Norriedisease, Prostatitis, Osteoarthritis, Osteoporosis, RheumatoidArthritis, Sickle Cell Anemia, heart disease, spinal and nerve relateddiseases and disorders, cancers (including for example but not limitedto leukemias and lymphomas), other injuries induced by trauma, orregeneration of tissue post-resectioning.

An exemplary method of inducing de-differentiation of human somaticcells is to 1) plate the cells on gelatin (e.g., plate 30,000 cells ongelatin in a 6-well plate); 2) at about 24 hours later, introduceexogenous factors such as Oct4, Sox2 and Klf4 into the cells (e.g., byviral transduction, transfection, or introduction of the proteins); 3)at 2 days post infection, switch the cells to human ES medium containing10 ng/ml bFGF. Transfection mixes consisting of 50 or 100 nM of thesmall RNA mimic and the transfection reagent Dharmafect1 are added tothe cells on days 3 and 10 post infection. The culture medium can bechanged every other day. Colonies typically appear between 2 to 4 weekspost infection. Colonies can be picked onto irradiated MEFs and passagedaccording to standard practices used for passaging human ES cells. Inembodiments where administration of a re-programmed human cell to humansis contemplated, “xeno free” or “humanized” conditions as known topersons of ordinary skill in the art can be used to culture the cells.

When embryonic stem cells (ESCs) differentiate, they must both silencethe ESC self-renewal program as well as activate new tissue specificprograms. In the absence of DGCR8 (Dgcr8−/−), a protein required formicroRNA (miRNA) biogenesis, mouse ESCs are unable to silenceself-renewal. Here, we also report that the introduction of let-7miRNAs, a family of miRNAs highly expressed in somatic cells, cansuppress self-renewal in Dgcr8−/−, but not wild-type ESCs. Introductionof ESC cell cycle regulating (ESCC) miRNAs into the Dgcr8−/− ESCs,blocks the capacity of let-7 to suppress self-renewal. Profiling andbioinformatic analyses show that let-7 inhibits while ESCC miRNAsindirectly activate numerous self-renewal genes. Furthermore, inhibitionof the let-7 family promotes de-differentiation of somatic cells toinduced pluripotent stem (iPS) cells. Together, these findings show howthe ESCC and let-7 miRNAs act through common pathways to alternativelystabilize the self-renewing versus differentiated cell fates.

Our findings show that the let-7 and ESCC miRNA families have opposingeffects on ESC self-renewal. Without being held to theory, we believethat they act in self-reinforcing loops to maintain the ESCself-renewing versus differentiated cell states (FIG. 14 b). In theself-renewing state, ESCC miRNAs would then indirectly increaseexpression of Lin28 and cMyc. Lin28 functions to block the maturation oflet-7 (Rybak, A. et al. Nat Cell Biol 10:987-93 (2008); Viswanathan, S.R. et al., Science 320:97-100 (2008); Heo, I. et al. Mol Cell 32:276-84(2008); Newman, M. A. et al., RNA 14:1539-49 (2008)). Therefore, theESCC miRNAs prevent co-expression of let-7 miRNAs. Additionally, withoutbeing bound to theory, we believe the ESCC-induced upregulation of cMycforms a positive feedback loop in which cMyc and nMyc, along withPou5fl/Oct4, Sox2, and Nanog, bind and activate expression of the ESCCmiRNAs in the miR-290 miRNA cluster (Judson, R. et al., Nat Biotech(2009); Marson, A. et al. Cell 134:521-33 (2008)). As ESCsdifferentiate, Pou5fl/Oct4, Sox2, and Nanog are then downregulated,resulting in the loss of ESCC and Lin28 expression. With the loss ofLin28, mature let-7 rapidly would increase. This increase in let-7 wouldthen be enhanced by a positive feedback loop in which let-7 suppressesits own negative regulator Lin28. In the differentiated state,downregulation of Myc activity by let-7 would prevent co-expression ofthe ESCC miRNAs. Furthermore, let-7 inhibits downstream targets ofPou5fl/Oct4, Sox2, Nanog, and Tcf3 to stabilize the differentiatedstate. Sa114, like Myc and Lin28, is positively regulated by the ESCCfamily and negatively regulated by let-7 family. Decreases in Myc,Sa114, and Lin28 all promote ESC differentiation (Lim, C. Y. et al. CellStem Cell 3:543-54 (2008); Zhang, J. et al. Nat Cell Biol 8:1114-23(2006); Heo, I. et al. Cell 138:696-708 (2009); Cartwright, P. et al.Development 132:885-96 (2005)).

In this model, the function of let-7 in repressing the self-renewingstate is restricted to cells that do not express high levels of ESCCmiRNAs. In fact, our model suggests that let-7 and ESCC miRNAs are notnormally co-expressed at high levels. For this reason, we propose thatthe let-7 family does not function to initiate differentiation, butrather the antagonism between the let-7 and ESCC families stabilizes theswitch between self-renewal and differentiation. Consistent with thismodel, the introduction of either ESCC miRNAs (Judson, R. et al., NatBiotech (2009)) or let-7 inhibitors into somatic cells promotes theirde-differentiation into iPS cells. Additionally, the ESCC and let-7miRNAs make up a preponderance of the miRNAs in self-renewing ESCs andsomatic cells respectively (Marson, A. et al. Cell 134:521-33 (2008)),supporting a major role in influencing these alternative cell fates.

Other miRNAs have been reported to target the ESC transcriptionalnetwork (Tay, Y. M. et al. Stem Cells 26:17-29 (2008); Tay, Y. et al.,Nature 455:1124-8 (2008); Xu, N. et al., Cell 137:647-658 (2009)).Unlike the let-7 family, these other miRNAs have a more limited tissuedistribution (Landgraf, P. et al. Cell 129:1401-1414 (2007); Chen, C. etal. Mamm. Genome 18:316-327 (2007)), suggesting that they may suppressself-renewal during differentiation along specific developmentalpathways. Alternatively, these miRNAs may be involved in the early andtransient stages of ESC differentiation while the let-7 miRNAs areinvolved in stabilizing the resulting differentiated cell fate. miRNAsrelated to the ESCC family (miR-17, miR-20, miR-93, and miR-106) andlet-7 miRNAs play analogous roles in cancer with the ESCC related miRNAspromoting and the let-7 miRNAs inhibiting cancer growth (Mendell, J. T.Cell 133:217-222 (2008); Bussing, I. et al., Trends in MolecularMedicine 14:400-409 (2008)). Thus, we contemplate that these miRNAs canact through similar opposing pathways in cancer.

A “target gene” refers to any gene suitable for regulation ofexpression, including both endogenous chromosomal genes and transgenes,as well as episomal or extrachromosomal genes, mitochondrial genes,chloroplastic genes, viral genes, bacterial genes, animal genes, plantgenes, protozoal genes and fungal genes.

A “microRNA” or “miRNA” refers to a nucleic acid that forms asingle-stranded RNA, which single-stranded RNA has the ability to alterthe expression (reduce or inhibit expression; modulate expression;directly or indirectly enhance expression) of a gene or target gene whenthe miRNA is expressed in the same cell as the gene or target gene. Inone embodiment, a miRNA refers to a nucleic acid that has substantial orcomplete identity to a target gene and forms a single-stranded miRNA. Insome embodiments miRNA may be in the form of pre-miRNA, wherein thepre-miRNA is double-stranded RNA. The sequence of the miRNA cancorrespond to the full length target gene, or a subsequence thereof.Typically, the miRNA is at least about 15-50 nucleotides in length(e.g., each sequence of the single-stranded miRNA is 15-50 nucleotidesin length, and the double stranded pre-miRNA is about 15-50 base pairsin length). In some embodiments the miRNA is 20-30 base nucleotides. Insome embodiments the miRNA is 20-25 nucleotides in length. In someembodiments the miRNA is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleotides in length. (see, Griffiths-Jones S, Saini H K, van Dongen S,Enright A J., NAR 2008 36 (Database Issue):D154-D158; Griffiths-Jones S,Grocock R J, van Dongen S, Bateman A, Enright A J., NAR 2006 34(Database Issue):D140-D144; Griffiths-Jones S. NAR 2004 32 (DatabaseIssue):D109-D111; and Ambros V, Bartel B, Bartel D P, Burge C B,Carrington J C, Chen X, Dreyfuss G, Eddy S R, Griffiths-Jones S,Marshall M, Matzke M, Ruvkun G, Tuschl T. RNA 2003 9(3):277-279).

A given miRNA sequence includes both the human and murine homologues ororthologs having structural and functional similarity to the referencedmiRNA. The term, homolog applies to the relationship between genesseparated by the event of speciation (see ortholog) or to therelationship between genes separated by the event of genetic duplication(see paralog). Orthologous miRNAs are miRNAs in different species thatare similar to each other because they originated from a commonancestor. Homologous sequences are similar sequences which share acommon ancestral DNA sequence or which would have been expected to sharesuch given their high degree of sequence identity. Accordingly, in someembodiments, the ortholog or homologue is any sequence which differsfrom the sequence of the referenced miRNA by at most one, two or threenucleic acid residues.

An inhibitor of a miRNA can be an antisense nucleic acid or siRNA whichis complementary to or shares substantial identity with the miRNA andcan block the function of the miRNA.

“Substantial identity” refers to a sequence that hybridizes to areference sequence under stringent conditions, or to a sequence that hasa specified percent identity over a specified region of a referencesequence.

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization.

Exemplary stringent hybridization conditions can be as following: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS,incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. ForPCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealingphase lasting 30 sec.-2 min., and an extension phase of about 72° C. for1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al. (1990) PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y.).

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology, ed. Ausubel, et al.

The terms “substantially identical” or “substantial identity,” in thecontext of two or more nucleic acids or polypeptide sequences, refer totwo or more sequences or subsequences that are the same or have aspecified percentage of amino acid residues or nucleotides that are thesame (i.e., at least about 60%, preferably 65%, 70%, 75%, preferably80%, 85%, 90%, or 95% identity over a specified region), when comparedand aligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection. Thisdefinition, when the context indicates, also refers analogously to thecomplement of a sequence. Preferably, the substantial identity existsover a region that is at least about 6-7 amino acids or 25 nucleotidesin length, or more preferably over a region that is 50-100 amino acidsor nucleotides in length, or the entire length. Preferable percentidentities are at least 65%, 70%, 75%, preferably at least 80%, 85%,90%, or 95% identity over the specified region

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions of a sequence. Thesegment can encompass an entire referenced sequence or be selected fromthe group consisting of from 10 to 600, 10 to 30, 20 to 600, about 50 toabout 200, or about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 1995supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always>0) and N (penalty score for mismatchingresidues; always<0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The phrases “regulating expression of a target gene” or “regulation ofgene expression” refer to the ability of a miRNA of the invention toregulate expression of the target gene. In some embodiments, generegulation can include targeting of mRNAs for cleavage. In someembodiments gene regulation can include translational repression. Toexamine the extent of gene regulation, samples or assays of the organismof interest or cells in culture expressing a particular construct arecompared to control samples lacking expression of the miRNA. Controlsamples (lacking construct expression) are assigned a relative value of100%. Inhibition of expression of a target gene is achieved when thetest value relative to the control is about 90%, preferably 50%, morepreferably 25-0%. Suitable assays include, e.g., examination of proteinor mRNA levels using techniques known to those of skill in the art suchas dot blots, northern blots, in situ hybridization, ELISA,immunoprecipitation and enzyme function, as well as phenotypic assaysknown to those of skill in the art.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), digoxigenin, biotin, luciferase, CAT, betagalactosidase, GFP, or haptens and proteins which can be madedetectable, e.g., by incorporating a radiolabel into the peptide or usedto detect antibodies specifically reactive with the peptide.

“Biological sample” includes tissue; cultured cells, e.g., primarycultures, explants, and transformed cells; cellular extracts, e.g., fromcultured cells, tissue, embryos, cytoplasmic extracts, nuclear extracts;blood, etc. Biological samples include sections of tissues such asbiopsy and autopsy samples, and frozen sections taken for histologicpurposes. A biological sample, including cultured cells, is typicallyobtained from a eukaryotic organism, most preferably a mammal such as aprimate, e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g.,guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish. iPS cellscan be derived from any biological sample, including but not limited tothose listed above.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

A particular nucleic acid sequence also implicitly encompasses “splicevariants.” Similarly, a particular protein encoded by a nucleic acidimplicitly encompasses any protein encoded by a splice variant of thatnucleic acid. “Splice variants,” as the name suggests, are products ofalternative splicing of a gene. After transcription, an initial nucleicacid transcript may be spliced such that different (alternate) nucleicacid splice products encode different polypeptides. Mechanisms for theproduction of splice variants vary, but include alternate splicing ofexons. Alternate polypeptides derived from the same nucleic acid byread-through transcription are also encompassed by this definition. Anyproducts of a splicing reaction, including recombinant forms of thesplice products, are included in this definition.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The term “autologous” when used herein designates host derived andtransplanted re-inserted, re-administered or returned to the host fromwhich the nucleic acid, protein, cell or tissue was derived. Autologouscan refer to nucleic acids, proteins, cells, or tissues derived from ahost and transplanted, re-inserted, re-administered or returned to thehost from which the nucleic acids, proteins or cells were derived.

The term “introducing” when used in the context of “introducing” aphysiologically relevant miRNA into a cell refers to any of thewell-known procedures for introducing foreign nucleotide sequences intohost cells may be used. These include the use of calcium phosphatetransfection, polybrene, protoplast fusion, electroporation, biolistics,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook et al., supra). It is only necessary that the particulargenetic engineering procedure used be capable of successfullyintroducing at least one miRNA into the host cell.

A variety of different types of cells can be utilized for the methods ofthe present invention. Cells that may express a microRNAs of theinvention can include, e.g., fibroblast cells. The cells can be any ofthe cells typically utilized in generating cells that harbor recombinantnucleic acid constructs. Exemplary cells include, but are not limited tocells such as COS, CHO, HeLa, 293T and mouse embryonic fibroblasts(MEFs). Cell types utilized for the methods of the present invention canalso include cells from tissue samples including but not limited toblood, bone, brain, kidney, muscle, spinal cord, nerve, endocrinesystem, uterine, ear, foreskin, liver, intestine, bladder or skin. Thecells can include neural cells, lymphocytes, epidermal cells, isletcells, intestinal cells or fibroblasts. The cells of the presentinvention can be autologous or heterologous cells. The cells useful forthe methods of the present invention can include animal cells. In someembodiments the cells are mammalian. In some embodiments the cell arefrom rodents or primates. In some embodiments the cells are mouse cells.In some embodiments the cells are human cells. In some embodiments thecells are diseased human cells, such as cancer cells. Cancer cells caninclude but are not limited to breast, prostate, liver, bladder, brain,blood or bone cancer cells.

The term “cell reprogramming” refers to altering the natural state ofthe cell such that the cell becomes pluripotent and is capable ofdividing and differentiating into any cell type other than embryoniccells. Cellular reprogramming can include inducing pluripotency in orde-differentiation of the cell. Altering cell reprogramming can alsorefer to enhancing the level of pluripotency or de-differentiation thathas been induced by an agent other than a microRNA. Pluripotent ormultipotent cells, also called stem cells, have the ability to divide(self-replicate or self-renew) or differentiate into multiple differentphenotypic lineages for indefinite periods; in some cases throughout thelife of the organism. Under specific conditions, or in the present ofoptimal regulatory signals, pluripotent cells can differentiate andtransform themselves into many different cell types that make up theorganism. Pluripotent or multipotent cells may be distinguishable fromprogeny daughter cells by such traits as BrdU retention and physicallocation/orientation in the villus microenvironment, or any othermethods well know to those of skill in the art. Multipotential orpluripotential stem cells possess the ability to differentiate intomature cells that have characteristic attributes and specializedfunctions, such as hair follicle cells, blood cells, heart cells, eyecells, skin cells, or nerve cells. A stem cell population is apopulation that possesses at least one stem cell. When pluripotent stemcells are derived from a non-pluripotent cell, such as for example asomatic cell, they are termed induced pluripotent stem cells (iPS oriPSCs). Cell reprogramming can further include partialde-differentiation to a closely related cell or cell type. Cellreprogramming can also include trans-differentiation.Trans-differentiation is defined as the conversion of one differentiatedcell type into another, such as for example conversion of exocrine cellsinto beta-islet-like cells. (See, e.g., Blelloch, et al., Short cut tocell replacement, Nature, 455:604-605 (2008).)

The term “degenerative disease” refers to any disease wherein anindividual would benefit from treatment with a pluripotent cell.Degenerative diseases include disease in which the function or structureof the affected tissue an/or organs progressively deteriorate over time.Examples of degenerative diseases include but are not limited toParkinson's Disease, Alzheimer's Disease, skin grafts, MuscularDystrophy, Amyotrophic Lateral Sclerosis (ALS) (e.g., Lou Gehrig'sDisease), Multiple system atrophy, Niemann Pick disease,Atherosclerosis, Progressive supranuclear palsy, cancer, metabolicdiseases (including for example but not limited to Tay-Sachs Disease),Diabetes, Heart Disease, Inflammatory Bowel Disease (IBD), Norriedisease, Prostatitis, Osteoarthritis, Osteoporosis, RheumatoidArthritis, Sickle Cell Anemia, heart disease, spinal and nerve relateddiseases and disorders, spinal and nerve related injuries, cancers(including for example but not limited to leukemias and lymphomas),other injuries induced by trauma, as well as regeneration of tissuepost-resectioning.

In some embodiments, the invention provides a method of inducingpluripotency in an isolated cell by introducing an inhibitor of let-7 oran inhibitor of an miRNA of examples 5 and 6 into the cell, wherein theinhibitor (ii) affects cell reprogramming to induce pluripotency in thecell. In some further embodiments, a physiologically relevant miRNA isfurther introduced into the cell, wherein the relevant miRNA: (i)contains the seed sequence AAGUGCU (SEQ ID NO: 15) or AAAGUGC (SEQ IDNO: 16) or AAGUGC and (ii) affects cell reprogramming to inducepluripotency in the cell. For instance, the relevant miRNA can be amember of the embryonic stem cell cycle (ESCC) regulating miR-290cluster, 302 cluster, 17-92 cluster, 106a, and 370 family of humanmicroRNAs. In some embodiments, the relevant miRNA is a human or mouseor mammalian miRNA. The cell can also be a human cell. The relevantmiRNA can also be (i) 80% or more identical to one of miR-291-3p (SEQ IDNO:1), miR-294 (SEQ ID NO:2), miR-295 (SEQ ID NO:3), miR-302d (SEQ IDNO:4), miR-292-3p (SEQ ID NO:5), hsa-mir-302a (SEQ ID NO:8),hsa-miR-302b (SEQ ID NO:9), hsa-miR-302c (SEQ ID NO:10), hsa-miR-302d(SEQ ID NO: 1), hsa-miR-372 (SEQ ID NO:13), hsa-miR-373 (SEQ ID NO:14),hsa-miR-17 (SEQ ID NO:17), hsa-miR-20a (SEQ ID NO:18), hsa-miR-20b (SEQID NO:19), hsa-miR-93 (SEQ ID NO:20), hsa-mir-106a (SEQ ID NO:21), orhsa-mir-106b (SEQ ID NO:22); and contain the seed sequence AAGUGCU (SEQID NO:15) or AAAGUGC (SEQ ID NO:16) or AAGUGC; or be a miR-290 clustermember which is one or more of miR-291-3p (SEQ ID NO: 1), miR-294 (SEQID NO:2) miR-295 (SEQ ID NO:3), miR-302d (SEQ ID NO:4), miR-292-3p (SEQID NO:5), hsa-mir-302a (SEQ ID NO:8), hsa-miR-302b (SEQ ID NO:9),hsa-miR-302c (SEQ ID NO: 10), hsa-miR-302d (SEQ ID NO: 11), hsa-miR371-5(SEQ ID NO:12), hsa-miR-372 (SEQ ID NO:13), or hsa-miR-373 (SEQ IDNO:14). In some embodiments, the effect on reprogramming is to enhancereprogramming (e.g., the enhancement of cell reprogramming includes orconstitutes enhancement of pluripotency or de-differentiation). ThemiRNA can also be an miRNA of Example 3 or Example 4 or their humanorthologs. In some embodiments of any of the above, the cell wasadministered Oct4, Sox2, and Klf4 (OSK) or transfected so as to expressOct4, Sox2, and Klf4 (OSK). Isolated cell populations and cell culturesobtained by these methods are also contemplated wherein th cell(s)contain(s) one or more miRNAs containing the seed sequence AAGUGCU (SEQID NO:15) or AAAGUGC (SEQ ID NO: 16) or AAGUGCU. Methods of treating anindividual with a degenerative disease comprising administering to anindividual a cell obtained by these methods are also contemplated by theinvention. The degenerative disease can be one or more of Parkinson'sDisease, Alzheimer's Disease, skin grafts, Muscular Dystrophy,Amyotrophic Lateral Sclerosis (ALS) (e.g., Lou Gehrig's Disease),Multiple system atrophy, Niemann Pick disease, Atherosclerosis,Progressive supranuclear palsy, cancer, metabolic diseases (includingfor example but not limited to Tay-Sachs Disease), Diabetes, HeartDisease, Inflammatory Bowel Disease (IBD), Norrie disease, Prostatitis,Osteoarthritis, Osteoporosis, Rheumatoid Arthritis, Sickle Cell Anemia,heart disease, spinal and nerve related diseases and disorders, cancers(including for example but not limited to leukemias and lymphomas),other injuries induced by trauma, or regeneration of tissuepost-resectioning.

The preferred miRNA of Examples 3 and 4 for use according to the variousembodiments and aspects of the invention are those listed in thoseexamples which provide, as set forth in the tables of these examples, anaverage difference in effect which is at least 2-, 3-, 4-, 5-, 6-fold,or even 10-fold different from the values for mock treated cells.Preferred miRNA of Example 5 for use according to the variousembodiments and aspects of the invention are those listed in the Tableof Ex. 5 which provide an average difference in effect which is at least½, ⅓, ¼, ⅕, ⅙ or even 1/10 of the values for mock treated cells.

EXAMPLES Example 1 Embryonic Stem Cell Specific microRNAs PromoteInduced Pluripotency

This work has now been published (see, Judson, R. et al., Nat Biotech27(5):459-461 (2009) which is incorporated herein by reference in itsentirety).

miRNAs Promote Induction of Pluripotency

The miRNAs miR-291-3p, miR-294, or miR-295 along with retrovirusesexpressing Oct4, Sox2, and Klf4 (OSK) were introduced into mouseembryonic fibroblasts (MEFs) (See, e.g., Takahashi, K. & Yamanaka, S.Induction of pluripotent stem cells from mouse embryonic and adultfibroblast cultures by defined factors Cell 126:663-676 (2006)). TheMEFs carried two reporters: an Oct4-GFP reporter that activates GFP withthe induction of pluripotency and ubiquitous expression of aJ-galactosidase/neo fusion from the Rosa26 locus. (See, e.g., Blelloch,R., Venere, M., Yen, J. & Ramalho-Santos, M. Generation of inducedpluripotent stem cells in the absence of drug selection Cell Stem Cell1:245-247 (2007)). MiRNAs were introduced on days 0 and 6 post-infectionby transfection of synthesized double-stranded RNAs that mimic theirmature endogenous counterparts. This method transiently recapitulatesES-like levels of the miR-290 cluster miRNAs (data not shown).

OSK plus miR-291-3p, miR-294, or miR-295 consistently increased thenumber of Oct4-GFP+ colonies as compared to controls transduced with OSKplus transfection reagent (FIG. 1 a). The miR-294 mimic showed thegreatest effects, increasing efficiency from 0.01-0.05% to 0.1-0.3% oftransduced MEFs. Introduction of a chemically synthesized miR-294pre-miRNA similarly enhanced reprogramming (FIG. 4). Two other membersof the miR-290 cluster that are not ESCC miRNAs, miR-292-3p and miR-293,did not increase colony number (FIG. 1 a). The ESCC miRNAs share aconserved seed sequence, which largely specifies target mRNAs (FIG. 1b). MiR-302d, a member of another miRNA cluster that has the same seedsequence also enhanced reprogramming (FIGS. 1 b&c). Mutation of the seedsequence in miR-294 blocked the increase in colony number (FIGS. 1 b&c).In summary, together with Oct4, Sox2, and Klf4, the ESCC miRNAs andrelated miRNAs with a common seed sequence (AAGUGCU; SEQ ID NO:15)promote the de-differentiation of fibroblasts into Oct4-GFP+ES cell-likecolonies.

Effect of Mixtures of miRNAs on Induction of Pluripotency

Consistent with previous observations that ESCC miRNAs act redundantly(Wang, Y. et al. Embryonic stem cell-specific microRNAs regulate theG1-S transition and promote rapid proliferation. Nat Genet 40, 1478-1483(2008)), mixes of the different ESCC miRNAs did not further enhancereprogramming efficiency (FIG. 5).

Studies Regarding miR-294 miRNA

Therefore, further studies focused on miR-294. Increasing doses ofmiR-294 further enhanced Oct4-GFP+ colony formation and the Oct4-GFP+cellular fraction (FIG. 1 d & FIG. 6). At the highest doses, miR-294increased the number of colonies to approximately 75 percent of thatachieved with OSK and cMyc (OSKM) (0.4-0.7% of starting MEFs) (FIG. 1d). Addition of miR-294 mimic increased the kinetics of OSKreprogramming to rates comparable to OSKM reprogramming (FIG. 7 a).Transfection of miR-294 did not further enhance the reprogrammingefficiency of any other three-factor combination or OSKM (FIG. 1 c &FIG. 7 b). Therefore, miR-294 substituted for, but did not furtherenhance, cMyc's contribution to reprogramming efficiency.

ES-like Oct4-GFP+ colonies induced by OSK and miR-294 (miR-294-iPS) wereexpanded and verified as induced pluripotent stem (iPS) cells.MiR-294-iPS lines expressed endogenous Oct4, Sox2, and Klf4, whileretrovirus expression was silenced (FIGS. 1 e & f). Colonies showed anES-like morphology and stained positively for the ES cell markers, Nanogand SSEA-1 (FIG. 8 a). The cell lines had normal karyotypes andefficiently induced teratoma formation with differentiation down allthree germ layers (FIG. 8 b & FIG. 9 a-c). Injection of miR-294-iPScells into blastocysts resulted in high-grade chimeras, withcontribution of donor iPS cells to all three germ layers, and to germline (FIGS. 1 g-h & FIG. 8 c).

Mechanisms of miRNA Substitutes for cMyc Reprogramming

The mechanism for how ESCC miRNAs substitutes for cMyc in reprogrammingis not entirely clear. However, bioinformatic analysis of ES ChIP-seqdata (Chen, X. et al. Integration of external signaling pathways withthe core transcriptional network in embryonic stem cells. Cell 133,1106-1117 (2008)) showed that both c-Myc and n-Myc bind to the promoterregion of the miR-290 cluster (FIG. 2 a). Oct4, Sox2 and Nanog have alsobeen reported to bind the promoter of the miR-290 cluster (Marson, A. etal. Connecting microRNA genes to the core transcriptional regulatorycircuitry of embryonic stem cells. Cell 134, 521-533 (2008)).Transduction of cMyc, Oct4, Sox2, or Klf4 expressing retrovirusindividually failed to induce expression of the miR-290 cluster infibroblasts (FIG. 2 b). Analysis of ChlP-seq data (Mikkelsen, T. S. etal. Genome-wide maps of chromatin state in pluripotent andlineage-committed cells. Nature 448, 553-560 (2007)) for differenthistone modifications (FIG. 2 c) showed that the miR-290 promoter isH3K27 methylated in MEFs, a modification associated with transcriptionalsilencing. In contrast, the promoter is H3K4 methylated in ES cells, amodification associated with transcriptional activity. Therefore, thesetranscription factors likely can only induce the expression of themiR-290 cluster as cells replace promoter-associated H3K27 with H3K4methylation during the reprogramming process. Indeed, with OSKMtransduction, miR-294 was robustly activated late in the reprogrammingprocess, similar to the reported timing for expression of endogenousOct4, and other critical members of the core ES machinery (FIG. 2 d)(Stadtfeld, M., Maherali, N., Breault, D. T. & Hochedlinger, K. Definingmolecular cornerstones during fibroblast to iPS cell reprogramming inmouse. Cell Stem Cell 2, 230-240 (2008); Brambrink, T. et al. Sequentialexpression of pluripotency markers during direct reprogramming of mousesomatic cells. Cell Stem Cell 2, 151-159 (2008)). These data suggestthat miR-294 is downstream of cMyc, but requires epigenetic remodelingfor expression.

The downstream effects of the ESCC miRNAs versus cMyc on thereprogramming process were not identical. Unlike cMyc, miR-294 did notpromote proliferation of MEFs early in the reprogramming process (FIG. 2e). Furthermore, as previously reported, approximately 80% of the OSKMcolonies failed to express Oct4-GFP and lacked ES-like morphology (FIGS.2 f & g) (Nakagawa, M. et al. Generation of induced pluripotent stemcells without Myc from mouse and human fibroblasts. Nat Biotechnol 26,101-106 (2008)). In contrast, OSK+miR-294 produced a predominantlyuniform population of ES-like GFP+ colonies. The Oct4-GFP-colonies wereinduced by cMyc, not inhibited by miR-294, as the introduction of bothproduced a similar number of GFP-, non-ES-like colonies as cMyc alone(FIG. 2 g). Finally, when cells were injected into immunodeficient miceto produce teratomas, more than a third of the teratomas resulting fromcMyc-iPS cells invaded into the underlying body wall, while none ofteratomas resulting from miR-294-iPS cells did so (FIG. 9 b&c). Thesefindings show that while miR-294 can substitute for cMyc to enhancereprogramming, its effects on the cell population are not identical.

In summary, our data show that miRNAs can replace cMyc in promoting thede-differentiation of somatic cells into induced pluripotent stem cells.An exciting possibility is that other small RNAs could replaceadditional factors, which together with other approaches may eventuallysubstitute for the use of introduced DNA elements. Additionally, furtheranalysis of the targets of these miRNAs may offer valuable insights intothe mechanism of reprogramming. The ESCC miRNAs are highly expressed inES cells where they promote progression through the ES cell cell cycle,by accelerating the transition through the G1/S restriction point Wang,Y. et al. Embryonic stem cell-specific microRNAs regulate the G1-Stransition and promote rapid proliferation. Nat Genet 40, 1478-1483(2008)). Their expression is downregulated with ES cell differentiationas the G1 phase of the cell cycle extends (Houbaviy, H. B., Murray, M.F. & Sharp, P. A. Embryonic stem cell-specific MicroRNAs. Dev Cell 5,351-358 (2003); Orford, K. W. & Scadden, D. T. Deconstructing stem cellself-renewal: genetic insights into cell-cycle regulation. Nat Rev Genet9, 115-128 (2008)). ESCC miRNAs have also been shown to induce theexpression of the de novo methyltransferases in ES cells, although howthis may promote self-renewal is unclear (Sinkkonen, L. et al. MicroRNAscontrol de novo DNA methylation through regulation of transcriptionalrepressors in mouse embryonic stem cells. Nat Struct Mol Biol 15,259-267 (2008); Benetti, R. et. al. A mammalian microRNA clustercontrols DNA methylation and telomere recombination via Rbl2-dependentregulation of DNA methyltransferases. Nat Struct Mol Biol 15, 998(2008)). As a target of Myc, the miR-290 cluster likely acts downstream,but only after erasure of silencing histone modifications within itspromoter. cMyc certainly has additional targets, reflected in thedifferences in outcomes between the introduction of cMyc and miR-294.

The ESCC miRNAs share a common seed sequence with a larger family ofsmall RNAs known to promote cellular proliferation (Takahashi, K. &Yamanaka, S. Induction of pluripotent stem cells from mouse embryonicand adult fibroblast cultures by defined factors. Cell 126, 663-676(2006)). This family includes “onco-miRs”, such as members of the miR-17cluster, miR-106, and miR-302 miRNAs (Mendell, J. T. miRiad roles forthe miR-17-92 cluster in development and disease. Cell 133, 217-222(2008); Voorhoeve, P. M. et al. A genetic screen implicates miRNA-372and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 124,1169-1181 (2006)). These miRNAs, like the ESCC miRNAs, may be acting byenhancing cell cycle progression and promoting de-differentiation of thecells. Such parallels between induced de-differentiation and cancer willbe an exciting area of future pursuit.

Materials and Methods

Cell Culture:

MEF isolation: E13.5 embryos were washed in HBSS. Heads and visceraltissues were removed, washed in fresh HBSS, briefly rinsed with 70%ethanol, then submerged in 0.05 mM trypsin/1 mM EDTA HBSS solution andincubated at 37° C. for 10 minutes. Embryos were pipetted repeatedly toaid in tissue dissociation, then added to MEF media containing 10% FBSand plated (passage 0). iPS lines were maintained in ES media+15%knock-out serum on irradiated MEF feeders or gelatin.

Retrovirus Infection:

The retroviral packaging vector pCL-ECO was transfected into 293T cellssimultaneously with pMXs vectors containing either Oct4, Sox2, Klf4, orcMyc cDNA using Fugene 6 (Roche) (Takahashi, K. & Yamanaka, S. Inductionof pluripotent stem cells from mouse embryonic and adult fibroblastcultures by defined factors. Cell 126, 663-676 (2006)). At 24 hours, themedia was changed, and at 48 hours, the media was collected, filtered(0.45

M), and frozen in aliquots at −80° C. Retrovirus was never thawed morethan once. To induce reprogramming, passage 3 Oct4-GFP MEFs were platedon gelatin at 3,000 cells per cm2. Virus-containing media was added 24hours later (Day 0). Cells were transfected with 1.6, 16 or 160 nMmicroRNA mimics (Dharmacon) with Dharmafect (Dharmacon) on daysindicated as according to manufacturers' protocol. MiR-294 seed sequencemutant contained mature sequence aaauuucuucccuuuugugugu. Media waschanged daily. Media was replaced with ES media+15% FBS on day 2, and ESmedia+15% knock-out serum replacement (Invitrogen) on day 6. Todetermine total cell number cells were trypsinized on day 7, counted,and passaged onto irradiated MEF feeders. GFP+ colonies were countedbetween days 10-12 and FACS sorted (BD FACSCalibur) on day 12. Percentefficiency was calculated as the fraction of colonies relative to totalMEFs infected. P-value of colony number was calculated using standardt-test. Individual iPS colonies were picked between days 10 and 15.

Immunohistochemistry:

iPS lines were grown in a 24-well plate, fixed with 4% paraformaldehydeand washed in 1×PBS with 0.1% Triton x-100 (PBT). PBT with 2% BSA and 1%goat-serum was used to block for one hour before addition of primaryantibodies against SSEAI (DSHB: MC-480) and Nanog (Abcam: ab21603),which were incubated overnight at 4° C. Cells were washed with PBT,blocked with PBT with 2% BSA and 10% goat-serum for 1 hour beforeaddition of secondary antibodies (Invitrogen: Alexa Fluor 594 goatanti-rabbit IgG and Biolegend: PE anti-mouse IgM).

Quantitative real-time PCR: Total RNA was isolated using TRizol(Invitrogen), polyadenylated (NEB), and DNase treated (Invitrogen),according to manufacturers' protocols. Reverse transcription wasperformed using the Superscript III kit (Invitrogen). Random hexamerswere used for mRNA analysis. Real-time quantitative PCR for mRNA wasconduced with SYBR Green PCR master mix (Applied Biosystems) accordingto the manufacturers' protocol using the following primer sets: Rex1,(gattgtggagccatacattgca, tgccgtagcctcgcttgt), Nanog(gctcagcaccagtggagtatcc, tccagatgcgttcaccagatag), Endogenous Oct4(tctttccaccaggcccccggctc, tgcgggcggacatggggagatcc) (Takahashi, K. &Yamanaka, S. Induction of pluripotent stem cells from mouse embryonicand adult fibroblast cultures by defined factors. Cell 126, 663-676(2006)), Endogenous Sox2 (tagagatagactccgggcgatga,ttgccttaaacaagaccacgaaa)1 (Takahashi, K. & Yamanaka, S. Induction ofpluripotent stem cells from mouse embryonic and adult fibroblastcultures by defined factors. Cell 126, 663-676 (2006)), Endogenous Klf4(gaattgtgtttcgatgatgc, tcgcttcctcttcctccgacaca), Lin28(agtctgccaagggtctggaa, cgctcactcccaatacagaaca), Exogenous Oct4(tctcccatgcattcaaactg, cttttattttatcgtcgacc), Exogenous Klf4(ccttacacatgaagaggcac, cttttattttatcgtcgacc), Exogenous Sox2(ctgcccctgtcgcacatgtg, cttttattttatcgtcgacc), cMyc(cagaggaggaacgagctgaagcgc, ttatgcaccagagtttcgaagctgttcg) and RPL7(gattgtggagccatacattgca, tgccgtagcctcgcttgt). Real-time quantitative PCRfor microRNAs was conducted using the Taqman approach as previouslydescribed (Tang, F., Hajkova, P., Barton, S. C., Lao, K. & Surani, M. A.MicroRNA expression profiling of single whole embryonic stem cells.Nucleic Acids Res 34, e9 (2006))

Teratoma Formation:

iPS lines were grown on irradiated MEFs or gelatin, trypsinized, andresuspended in PBS. One million iPS cells were injected subcutaneouslyper side in severe combined immunodeficient (SCID) mice (NCI-Frederick).Tumors were removed when they reached a size of 1-1.5 cm in longdiameter, fixed in 10% formalin, embedded in paraffin, sectioned, andH&E stained.

Blastocyst Injection and Chimera Formation:

Super-ovulation of B6D2F1/Cr females (NCI-Frederick) was induced viaPMSG injection (d0) and hCG injection (d2). On d2, females were crossedto B6D2F1/Cr males, oocytes were isolated on d3, washed in M2 media(Specialty Media) and grown in KSOM media (Specialty Media) for threedays. IPS cells were first karyotyped as previously described. 8-12cells were injected into cultured blastocysts, which were thentransplanted into the uteri of pseudo-pregnant Swiss-Webster females.Embryos were collected on E15 and stained as previously described.

Mir-290 Promoter Analysis:

Previously published ChIP-seq data for c-Myc, n-Myc, H3K4me3, andH3K27me3 were downloaded as fastq files and aligned to the mm9 (NCBIBuild 37.1) assembly of the mouse genome using Eland (GA Pipeline 1.0,Illumina). The mm9 assembly contains the mir-290 locus, which wasmissing from previous assemblies. Following alignment, peak scores wereassigned using the Findpeaks 3.1.9.2 algorithm. The peak scores werenormalized to the number of genome-mapping sequence reads.

Example 2 Let-7 and Esccs Regulate Self-Renewal

The let-7 miRNAs are broadly expressed across differentiated tissues(Landgraf, P. et al. Cell 129:1401-1414 (2007); Chen, C. et al. Mamm.Genome 18:316-327 (2007)) and are tightly regulated during ESCdifferentiation (Rybak, A. et al. Nat Cell Biol 10:987-93 (2008);Viswanathan, S. R. et al., Science 320:97-100 (2008); Heo, 1. et al. MolCell 32:276-84 (2008 Newman, M. A. et al., RNA 14:1539-49 (2008);Thomson, J. M. et al. Genes Dev 20:2202-7 (2006)). To test thehypothesis that let-7 miRNAs could rescue the capacity of Dgcr8−/− ESCsto silence ESC self-renewal when induced to differentiate, we introducedmimics of a representative let-7 family member, let-7c, into theDgcr8−/− ESCs (FIG. 10 a). Let-7c silenced the ESC self-renewal programeven when the ESCs were maintained in ESC culture conditions. Three daysafter treatment with let-7c, Dgcr8−/− cells downregulated ESC associatedmarkers including alkaline phosphatase activity, Pou5fl/Oct4immunofluorescence staining), and mRNA expression of Pou5fl/Oct4, Sox2,and Nanog (data not shown). Furthermore, the transfected cells showed adiminished capacity to reform ESC colonies in replating assays, afunctional test of ESC self-renewal capacity (FIG. 10 d, panel i).Similar effects were observed with the introduction of let-7a, let-7b,let-7d, and let-7g (FIG. 15) and these effects were observed over arange of concentrations, including levels normally found in moredifferentiated cell types (data not shown).

In contrast to the Dgcr8−/− ESCs, wild-type ESCs were resistant tolet-7c (FIG. 1 b-d, panel ii). This finding suggested that other miRNAsnormally expressed in wild-type ESCs inhibit let-7c-induced suppressionof self-renewal. The ESCC miRNAs are likely candidates as they make up amajority of miRNA molecules in mouse ESCs (Marson, A. et. al. Cell134:521-33 (2008); Calabrese, J. M., Proc. Natl. Acad. Sci. U.S.A104:18097-18102 (2007)), they are rapidly downregulated upondifferentiation coincident with the upregulation of mature let-7 (datanot shown), and they promote the ESC fate (Wang, Y. et al. Nat Genet40:1478-83 (2008); Judson, R. et al., Nat Biotech (2009); Benetti, R. etal., Nat Struct Mol Biol 15 (3):268-279 (2008); Sinkkonen, L. et al.,Nat Struct Mol Biol 15 (3):259-267 (2008)). Therefore, we introduced arepresentative member of this family, miR-294, to test if it could blocklet-7c-induced suppression of Dgcr8−/− ESC self-renewal. Three daysafter co-introduction of miR-294 and let-7c, Dgcr8−/− ESCs retainedalkaline phosphatase activity (data not shown), Pou5fl/Oct4immunofluorescence staining (FIG. 10 b, panel i), and mRNA expression ofPou5fl/Oct4, Sox2, and Nanog (FIG. 10 c, panel i). Furthermore, miR-294rescued the colony forming capacity of the Dgcr8−/− ESCs (FIG. 10 d,panel i). Control miRNAs (miR-294 with a seed mutation and other ESCexpressed miRNAs, miR-291a-5p and miR-130b, that do not contain the ESCCmiRNA seed sequence) did not antagonize the effects of let-7c (FIG. 10a-d) showing that miR-294's effect is not simply secondary tocompetition for RISC complexes. Other members of the ESCC familymiR-291a-3p, miR-291b-3p, and miR-295 were similarly able to block theeffects of let-7c (FIG. 16). These data indicate that the let-7 and ESCCfamilies of miRNAs have opposing roles in the maintenance of ESCself-renewal.

Targeting Through ORFs and 3'UTRs

The functional antagonism between let-7c and miR-294 on ESC self-renewalsuggested opposing roles for these miRNAs on downstream moleculartargets. To test this prediction, we sought to globally identify thesetargets using mRNA microarrays following the introduction of let-7c ormiR-294 into Dgcr8−/− ESCs. The introduction of the let-7c mimic led todownregulation of 693 and upregulation of 208 transcripts relative tomock treated cells with a false discovery rate (FDR) less than 5% (FIG.11 a, Table S1). Of the 693 downregulated transcripts, 294 contained alet-7c 7mer seed match in the 3'UTR, 287 contained a 7mer seed match inthe ORF, and 113 contained both 3'UTR and ORF seed matches (Table S1).The presence of these seed matches in the downregulated transcripts washighly enriched compared to the entire gene set (FIG. 11 b, FIG. S6a).Similarly, the introduction of miR-294 led to a large number ofupregulated and downregulated transcripts (FIG. 1 c, Table S1). Again,downregulated transcripts were enriched for seed matches in the 3'UTRand ORF. In contrast, upregulated transcripts were depleted for seedmatches in the 3'UTR and ORF (FIGS. 11 b&d). These findings suggest thatmiR-294 and let-7c functionally act through the down-regulation of manytargets by binding their ORF and/or 3'UTR.

Impact on ESC Transcriptional Network

To further investigate the mechanism for the opposing roles of let-7cand miR-294 on ESC self-renewal, we performed pathway analysis on themiRNA regulated transcript sets. Specifically, we searched for overlapsbetween the miRNA-regulated transcripts and genes identified bychromatin immunoprecipitation (ChIP) of pluripotency associatedtranscription factors (Marson, A. et al. Cell 134:521-33 (2008); Chen,X. et al. Cell 133:1106-17 (2008)). This analysis measures whether thereis any influence of the let-7 or ESCC miRNAs on the transcriptionfactors themselves (FIG. 12 a, i&ii) or the transcripts originating fromthe genes bound by the transcription factors (FIG. 12 a, iii).

In ESCs, two Myc family members—nMyc and cMyc—are highly expressed andhave largely overlapping ChIP target genes (Chen, X. et al. Cell133:1106-17 (2008)). cMyc has previously been identified as a let-7target in cancer cells (Kumar, M. S. et al., Nat Genet 39:673-677(2007)), and we find that nMyc is significantly downregulated by let-7cin our array data (not shown). Consistent with let-7 directly targetingthe Myc family, overlapping let-7c-regulated transcripts with Myc-boundgenes showed an enrichment of Myc target genes in thelet-7c-downregulated transcript set and a depletion in thelet-7c-upregulated transcript set (FIG. 12 b, Box I). Furthermore, theenrichment was independent of the presence of seed sequence matcheswithin the ORF or 3'UTR. This finding suggests that let-7 is actingdirectly through Myc (cMyc and/or nMyc) rather than through Myc'sdownstream target genes (FIG. 12 a, i).

Performing a similar analysis overlapping miR-294-regulated transcriptsand Myc target genes showed the exact opposite pattern as the analysiswith let-7c-regulated transcripts. There was a depletion for Myc targetsin the miR-294-downregulated transcript set and an enrichment in themiR-294-upregulated transcript set (FIG. 12 b, Box II). This patternsuggests that miR-294 upregulates Myc activity (FIG. 12 a, ii). Indeed,microarray data showed that miR-294 dramatically increased cMyc levels(data not shown). As miR-294 itself suppresses its downstream targets(FIG. 11 d), the upregulation of cMyc must be indirect, through anunknown intermediate repressor (FIG. 12 a, ii). These data show that thelet-7 and ESCC families of miRNAs have opposing effects on Myc activity.

Overlap of the let-7c-regulated transcripts with ChIP target genes forthe pluripotency transcription factors, Pou5fl/Oct4, Sox2, Nanog, andTcf3 once again showed an enrichment among let-7c-downregulatedtranscript set (FIG. 12 b, Box III). However, this enrichment waslimited to the downregulated transcripts with seed matches in their ORFor 3'UTR. These data suggest that rather than directly regulating thepluripotency transcription factors, let-7 targets transcriptsoriginating from the genes bound by them (FIG. 12 a, iii). This patternof enrichment is most clear for the ChIP target genes bound by Tcf3,cobound by Pou5fl/Oct4, Sox2, and Nanog, or bound by Chen et al.'spluripotency cluster (a group of targets bound by Pou5fl/Oct4, Sox2,Nanog, Smad1, and STAT3). The latter results agree with recent reportsshowing that genes bound by multiple pluripotency transcription factorsare more likely to be transcriptionally activated (Chen, X. et al. Cell133:1106-17 (2008); Kim, J. et al., Cell 132:1049-61 (2008)). There wasno enrichment in the overlap between the miR-294-regulated transcriptsand Pou5fl/Oct4, Sox2, Nanog, and Tcf3 bound genes. These data suggestthat let-7c inhibits downstream targets of these pluripotency factorswhile miR-294 has no obvious effects on either the transcription factorsthemselves or on their downstream targets.

Opposing Regulation of Myc, Lin28, and Sa114

Having discovered that Myc activity was alternatively downregulated andupregulated by let-7c and miR-294, we sought to identify other factorsthat might be similarly regulated by these miRNAs. Indeed, gene ontologyanalysis showed an enrichment for ESC enriched genes among thelet-7c-downregulated and miR-294-upregulated transcript sets (not TableS2). 88 transcripts were regulated in opposing directions by let-7c andmiR-294, of which 44 contained a let-7c seed match (data not shown).Notably, this set of transcripts included the well-known pluripotencygenes Lin28 and Sa114. Lin28 is an RNA binding protein that inhibitslet-7 processing (Rybak, A. et al. Nat Cell Biol 10:987-93 (2008);Viswanathan, S. R. et al., Science 320:97-100 (2008); Heo, I. et al. MolCell 32:276-84 (2008); Newman, M. A. et al., RNA 14:1539-49 (2008);Piskounova, E. et al. J Biol Chem 283:21310-4 (2008)), but nottransfected let-7 mimic (data not shown). Sa114 is a transcriptionfactor that promotes ESC self-renewal (Lim, C. Y. et al. Cell Stem Cell3:543-54 (2008); Wu, Q. et al. J Biol Chem 281:24090-4 (2006); Zhang, J.et al. Nat Cell Biol 8:1114-23 (2006)). These findings show that thelet-7 and ESCC families antagonistically regulate multiple genes withdescribed roles in ESC self-renewal.

To verify our genomic analysis, we performed qRT-PCR, Western analysis,and reporter assays for a subset of the genes. qRT-PCR confirmed theopposing effects of let-7c and miR-294 on Lin28, Sa114, nMyc, and cMycmRNA levels with a combination of the two miRNAs showing intermediatelevels (FIG. 13 a). Western analysis showed similar results (FIG. 13 b).Of note, cMyc protein was dramatically reduced in Dgcr8−/− versuswild-type ESCs and was brought back to wild-type levels with theintroduction of miR-294. MiR-294 had little effect on nMyc levels. Incontrast, let-7c had little effect on cMyc, yet dramatically reducednMyc levels. Therefore, the cumulative effect of the miRNAs on total Myc(cMyc+nMyc) protein levels followed a strong pattern of opposingregulation. Similarly, the miRNAs showed significant opposing effects onLin28 and Sa114 protein levels. Lin28 and cMyc are known targets oflet-7 (Rybak, A. et al. Nat Cell Biol 10:987-93 (2008); Kumar, M. S. etal., Nat Genet 39:673-677 (2007)), and luciferase assays confirmed thatnMyc and Sa114 are also direct targets (FIG. 13 c).

Considering that cMyc was dramatically reduced in Dgcr8−/− cells andthen increased with miR-294, we considered the possibility that the lossof cMyc alone could largely explain the sensitivity of Dgcr8−/− cells tolet-7-induced silencing of ESC self-renewal. To test this possibility,we generated and evaluated cMyc−/− ESCs. The loss of cMyc led todecreased expression of Pou5fl/Oct4 relative to the parental cell line(data not shown). Introduction of let-7c into the cMyc−/− cellsdecreased the expression levels of Sox2 and Nanog (data not shown).However, levels were not reduced to the same degree as seen with theintroduction of let-7c into Dgcr8−/− cells. These results indicate thatthe decrease of cMyc in Dgcr8−/− cells alone cannot explain thesensitivity of these cells to let-7-induced silencing of ESCself-renewal.

Inhibition of Let-7 Promotes De-Differentiation

Having identified a pro-differentiation function of the let-7 family ofmiRNAs, we hypothesized that inhibition of this miRNA family wouldenhance reprogramming of somatic cells to iPS cells. Indeed, Lin28,among other activities (Heo, I. et al. Cell 138:696-708 (2009); Xu, B.et al., RNA 15:357-361 (2009); Jones, M. R. et al. Nat. Cell Biol11:1157-1163 (2009); Polesskaya, A. et al. Genes Dev 21:1125-1138(2007)), inhibits let-7 biogenesis (Rybak, A. et al. Nat Cell Biol10:987-93 (2008); Viswanathan, S. R. et al., Science 320:97-100 (2008);Heo, I. et al. Mol Cell 32:276-84 (2008); Newman, M. A. et al., RNA14:1539-49 (2008)) and promotes de-differentiation of human somaticcells to iPS cells (Yu, J. et al. Science 318:1917-1920 (2007)).Reprogramming to iPS cells is typically achieved by the introduction ofvirally expressed Pou5fl/Oct4, Sox2, and Klf4 with or without Myc intosomatic cells such as mouse embryonic fibroblasts (MEFs). While Mycdramatically increases the efficiency of reprogramming, it is notessential (Nakagawa, M. et al. Nat. Biotechnol 26:101-106 (2008);Wernig, M. et al., Cell Stem Cell 2:10-12 (2008)). To test the impact oflet-7 family on reprogramming, we used a let-7 antisense inhibitor. Thisinhibitor was able to suppress multiple let-7 family memberssimultaneously (FIG. 17).

MEFs express high levels of mature let-7 (Marson, A. et al. Cell134:521-33 (2008)) and, therefore, these cells should be responsive toany pro-reprogramming effects of let-7 downregulation. We used Oct4::GFPtransgenic MEFs in order to quantify changes in reprogrammingefficiencies as Oct4::GFP is activated late in the reprogramming process(Stadtfeld, M. et al., Cell Stem Cell 2:230-240 (2008); Brambrink, T. etal. Cell Stem Cell 2:151-159 (2008)). MEFs were transduced withretroviral vectors expressing Pou5fl/Oct4, Sox2, Klf4, with or withoutcMyc on day 0 as well as transfected with let-7 or a control inhibitoron days 0 and 6. When 3 transcription factors were used (minus cMyc),let-7 inhibition increased the number of GFP positive colonies on day 10by 4.3 fold compared to mock whereas a control inhibitor had nosignificant effect (FIG. 14 a, left panel). In the presence of all 4transcription factors, let-7 inhibition resulted in a 1.75 fold increase(FIG. 14 a, right panel). Immunofluorescence confirmed expression ofNanog in reprogrammed cells (data not shown). Furthermore, the resultingiPS cells expressed endogenous pluripotency markers at levels similar towild-type ESCs and did not express the exogenously introduced factors(data not shown), as expected for fully reprogrammed cells(Hochedlinger, K. et al., Development 136 (4):509-523 (2009)). Theimpact of the let-7 inhibitor is not due to enhanced proliferation ofthe MEFs as there was actually a subtle decrease in proliferationfollowing transfection of either the let-7 or control inhibitor (datanot shown). These findings show that inhibition of let-7 family ofmiRNAs enhances the reprogramming of somatic cells. The finding that theenhancement was greater in absence of Myc is consistent with Mycactivity being one, but not the only important downstream target oflet-7 in stabilizing the somatic cell fate.

Methods Summary

Dgcr8−/− and wild-type V6.5 ESCs were cultured as previously described(Wang, Y. et al., Nat Genet 39:380-5 (2007)). miRNA mimics andinhibitors were obtained from ThermoFisher. mRNA profiling was performedon Affymetrix Mouse Gene 1.0 ST arrays. Bioinformatic analysis wasperformed using significance analysis of microarrays (SAM), R packages,and custom Python scripts. Reprogramming with Oct4-GFP MEFs wasperformed as previously described (Judson, R. et. al., Nat Biotech(2009)). See, also Melton et. al., Nature 463:621-626 (2010) which areincorporated herein by reference in their entirety with respect to themethods and results disclosed therein.

Tissue Culture, Transfection and Alkaline Phosphatase Staining.

ESC lines and culture conditions were previously described³. ESCs wereweaned off MEFs and maintained in MEF conditioned media. For ESCdifferentiation assays, 40,000 Dgcr8^(−/−) or 12,000 wild-type ESCs wereplated in gelatinized 12-well plates (or half the number of cells wereplated on 24-well plates) on day 0 in LIF media. On day 1, miRIDIANmiRNA mimics (Dharmacon, ThermoFisher) were transfected at aconcentration of 50 nM using Dharmafect1 (Dharmacon, ThermoFishcr)following the manufacturer's protocol. Media was changed daily. On thethird day after transfection, cells were either lysed in Trizol(Invitrogen) for qRT-PCR analysis or fixed in 4% paraformaldehyde (PFA)for alkaline phosphatase staining. Alkaline phosphatase staining wasperformed per the manufacturer's instructions (Vector Labs). iPS celllines were maintained in ESC media plus 15% knockout serum on irradiatedMEF feeders. Colony reformation assays were performed as previouslydescribed3. In brief, cells were exposed to miRNA mimics for 3 days thentrypsinized and counted. A defined number of cells were replated on MEFsto form colonies for 5-7 days. The efficiency of colony reformation wasdetermined by dividing the number of alkaline-phosphatase-positivecolonies by the number of cells plated. Some neural progenitor can begenerated by in vitro differentiation of ESCs as described previously(Okabe, S., Forsberg-Nilsson, K., Spiro, A. C., Segal, M. & McKay, R. D.Development of neuronal precursor cells and functional postmitoticneurons from embryonic stem cells in vitro. Mech. Dev. 59, 89-102(1996)).

ESC Derivation.

Timed matings were set up for c-myc f/f mice (Trumpp et al., Nature414:768-773 (2001). ESCs were derived from embryos isolated at embryonicday (E)3.5. These embryos were cultured on an irradiated MEF feederlayer in ESC media supplemented with 50 mM PD98059 (Buehr, M. & Smith,A. Genesis of embryonic stem cells. Phil. Trans. R. Soc. Lond. B 358,1397-1402 (2003)) and disassociated onto fresh feeders. ESCs were PCRgenotyped as previously described (Trumpp et al., Nature 414:768-773(2001). Aflox/flox line was grown out, infected with Ad5 Cre-IRES-GFPvirus, sorted by FACS, and plated back onto MEF feeders. c-myc2/2colonies were grown out and verified by PCR genotyping and westernblotting.

mRNAarrays.

qRT-PCR showed that mRNAlevels of a known let-7 target, Lin28, wasmaximally reduced 12 h after transfection before a large decline in Oct4and Nanog (data not shown). Therefore, we chose 12 h for all microarrayanalysis to minimize secondary effects of let-7c-induceddifferentiation. On day 0, 150,000 cells were plated in a 3.5-cm dish.miRIDIAN miRNA mimics (Dharmacon, ThermoFisher) were transfected at aconcentration of 50 nM in media in the absence of LIF. At 12 h aftertransfection cells were lysed in Trizol (Invitrogen) and RNA wasprepared according to the manufacturer's protocol. Affymetrix Mouse Gene1.0 ST arrays were probed by the Gladstone Genomics Core. Threebiological samples were assayed for each treatment. Data were analyzedby Affymetrix Expression Console software. The robust multichip analysis(RMA) algorithm was used to normalize the array signal across chips. SAMwas used to determine FDRcutoffs for significantly altered genes.

qRT-PCR Analysis.

RNA for all qRT-PCR analyses was prepared using Trizol (Invitrogen) andquantified on a Nanodrop Spectrophotometer (ThermoFisher). Five-hundrednanograms of RNA was DNase-treated using DNasel amplification grade(Invitrogen). For qRT-PCR of mRNAs, DNase-treated samples werereverse-transcribed using the Superscript III first-strand synthesissystem for RT-PCR (Invitrogen). qPCR reactions on resulting cDNAs wereperformed on either an ABI Prism 7100 or an ABI 7900HT (AppliedBiosystems). For miRNAs, qRT-PCR was performed either by using TaqManmiRNA assays (Applied Biosystems) or by polyadenylating the miRNAs andthen using a modified oligodT reverse transcription primer as describedpreviously (Shi, R. & Chiang, V. L. Facile means for quantifyingmicroRNA expression by realtime PCR. Biotechniques 39, 519-525 (2005)).

Lin28 and GFP Expression in 293T Cells.

Lin28 was cloned into an expression vector under the EF1α promoter andupstream of IRES Pac (puromycin resistance). A similarly constructed GFPexpression construct was previously generated. 293T cells weretransfected with 5 μg of each construct and selected with 0.6 μg/mlpuromycin for 12 days.

Luciferase Reporter Assays.

Constructs were produced as follows. The N-myc and Sa114 3'UTRs wereamplified from ESC cDNA and cloned into the NotI and XhoI sites inpsiCheck-2 vector (Promega). Mutant UTRs were generated by a two-stepPCR strategy with overlapping mutated PCR primers. Products of two PCRswith mutations were used in a second PCR reaction to generatefull-length mutated inserts that were cut and ligated into a cut emptyvector. For transfections, 8,000 Dgcr8/ESCs were plated in ESC media ina 96-well plate pretreated with 0.2% gelatin. The next day, miRIDIANmiRNA mimics (Dharmacon, ThermoFisher) were transfected with Dharmafect1(Dharmacon, ThermoFisher) following the manufacturer's protocol at aconcentration of 100 nM. Simultaneously, luciferase constructs weretransfected into ESCs at a concentration of 200 ng per well using FUGENE6 (Roche) transfection reagent following the manufacturers protocol. Thenext day, 14-18 h later, cells were lysed and luciferase assays wereperformed using a Dual-Luciferase Reporter Assay System (Promega) on asingle automatic injection Mithras (Berthold technologies) luminometerfollowing the manufacturer's protocol. Transfection of each constructwas performed in triplicate in each assay. Ratios of Renilla luciferasereadings to firefly luciferase readings were averaged for eachexperiment. Replicates performed on separate days were mean centeredwith the common readings from the individual days.

Seed Match Analysis.

Promoter (1,000 base pairs from the transcriptional start), 5'UTR, ORFand 3'UTRs for Ensembl Transcripts (mm9) and known genes (mm8) weredownloaded separately from the UCSC Genome Browser Table Browser. Seedmatch analysis was performed on these transcripts using a custom Pythonscript. 7-nucleotide seeds were defined as either 7mer-1A or 7mer-m8(Lewis, B. P., Burge, C. B. & Bartel, D. P. Conserved seed pairing,often flanked by adenosines, indicates that thousands of human genes aremicroRNA targets. Cell 120, 15-20 (2005)). Seed match results weremapped to Affymetrix IDs. A Python script was then implemented toeliminate redundant transcripts as transcripts often mapped 0.1:1 withAffymetrix IDs. The transcript with the most 7-nucleotide seed matcheswas chosen to produce a 1:1 transcript to Affymetrix ID mapping. Thismapping was done separately for the promoters, 5'UTRs, ORFs and 3'UTRs.In rare cases, duplicate Affymetrix IDs exist for the same gene. Thesewere retained in our analyses. Microsoft Access (Microsoft) was used togenerate list overlaps for analyses. P-values were calculated in FIGS.11 b, d with the number of seed matches per kb of transcript usingWilcoxon's rank sum test in R. P-values were calculated using a binary 0for no seed matches or 1 for a seed match using the hypergeometricdistribution function in R.

ChIP Target Overlap Analysis.

ChIP targets were downloaded as described in Melton et al, Nature463:621-626 (2010). Scripts were written to convert provided transcriptIDs to a non-redundant list of Affymetrix IDs. Microsoft Access(Microsoft) and custom Python scripts were used to perform comparisonsbetween gene lists and ChIP gene target lists. ChIP data describedpreviously was downloaded as an association score between any particulargene and the transcription factor of interest (Chen, X. et al. Cell133:1106-17 (2008)). These scores were used directly for enrichment. Forthe Oct4-, Sox2-Nanogbound group per Chen et al., any score above 0 wascounted as bound. For all data, enrichment for ChIP gene target sets inmiRNA-regulated gene sets was performed relative to all genes analysedto produce the miRNA-regulated gene sets (that is, all genes withAffymetrix IDs mapping to coding transcripts). The enrichments for anygiven ChIP target set were median normalized with all themiRNA-regulated genes sets in FIG. 12B. We performed this normalizationbecause both the ChIP targets of the transcription factors and themiRNAregulated gene sets in our analysis are enriched for more highlyexpressed genes. We get a similar pattern of results without thisnormalization, although all comparisons appear more highly enrichedowing to the expression levels (data not shown). Un-normalizedenrichment is defined as: (genes in overlap of miRNA-altered group andChIP group/all genes in miRNA-altered group)/(all genes in ChIPgroup/all genes used in analysis to generate miRNA altered groups).

Our enrichment analysis could yield several possible outcomes dependingon whether the miRNA targeted the transcription factor directly versustargeted transcripts downstream of the transcription factor. Thefollowing outcomes are presented in FIG. 12A. (1) If an miRNA directlytargets a specific transcriptional activator, this activator will bedownregulated, and thus its ChIP target genes will likewise tend to bedownregulated. This will result in an enrichment of ChIP target geneswithin the miRNA's downregulated gene set independent of there being aseed match in these targets. Similarly, the ChIP target genes should bedepleted in the miRNA's upregulated gene set (FIG. 12A, a). (2) If anmiRNA directly targets a transcriptional repressor, there would be theinverse outcome; that is, the ChIP target genes should be enriched inthe miRNA's upregulated gene set and depleted in the miRNA'sdownregulated gene set regardless of seed match (FIG. 3A, b). If anmiRNA targets an activating transcription factor's downstream targets,but not the transcription factor itself, ChIP target genes would beenriched in the downregulated gene set with a seed match but not withouta seed match. Furthermore, there should not be enrichment in theupregulated transcripts (FIG. 12A, c).

Gene Ontology.

Stem-cell-associated genes (genes upregulated in ESCs relative to brainand bone marrow) were generated from data described previously (Lewis etal., Cell 120:15-20 (2005) and were downloaded as a list from MySigDB.Enrichment of these stem cell associated genes in miRNA altered genesets was performed, and P-values were calculated by Fischer's exacttest.

Immunohistochemistry.

Cells were fixed with 4% PFA and washed twice in PBS with 0.1% TritonX-100 (PBT). PBT with 2% bovine serum albumin (BSA) and 1% goat-serumwas used to block for 1 h before the addition of primary antibodyagainst Oct4 (Santa Cruz, rabbit polyclonal, product sc-9081) or Nanog(Calbiochem, rabbit polyclonal, product sc-1000), which was incubatedovernight at 4° C. or at room temperature for approximately 2 h. Cellswere washed with PBT, blocked with PBT plus 2% BSA and 10% goat-serumnfor 1 h before addition of secondary antibodies (Alexa Fluor 488 goatanti-rabbit IgG, Invitrogen).

Western Blots.

On day 0, approximately 200,000 Dgcr8^(−/−) or 50,000 wild-type ESCswere plated in a 6-well plate. The next day miRIDIAN miRNA mimics(Dharmacon, ThermoFisher) were transfected at a concentration of 50 nM.Lysates were collected 2 days after transfection in EBC buffer (50 mMTris-HCl, pH 8.0, 120 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA) containing1× protcase inhibitor cocktail (Roche). Lysates were incubated at 4° C.for 45 min rocking then spun at 4° C. and approximately 20,000 g in atable-top centrifuge. Protein was quantified using a Bio-Rad proteinassay (Bio-Rad). Thirty micrograms of protein was resolved on an 8%SDS-PAGE gel. Proteins were transferred to Immobilon-FL (Millipore) andprocessed for immunodetection. Blots were scanned on a Licor OdysseyScanner (Licor). The actin antibody was used at a 1:1,000 dilution(Sigma, mouse monoclonal clone AC-40, A4700), the c-Myc antibody at1:500 (Epitomics, N-term rabbit monoclonal, 1472-1), the N-Myc antibodyat 1:500 (Calbiochem, mouse monoclonal, OP13), the Nanog antibody at1:1,000 (Abcam, rabbit polyclonal, ab21603), the Sa114 antibody at 1:500(Abcam, rabbit polyclonal, ab29112), and the Lin28 antibody at 1:1,000(Abcam, rabbit polyclonal, ab46020). Secondary infrared-dye antibodiesfrom Licor were used at 1:10,000. Data were exported from the LicorOdyssey as jpg and quantified using ImageJ software (NIH).

MEF Isolation.

E13.5 embryos from Oct4-GFP/Rosa-26-β-galactosidase transgenic crosseswere isolated by Caesarean section and washed in HBSS. Heads andvisceral tissues were removed. Remaining tissue was washed in freshHBSS, briefly rinsed with 70% ethanol, then submerged in 0.05 mMtrypsin/1 mM EDTA HBSS solution and incubated at 37° C. for 10 min.Tissue was pipetted repeatedly to aid in tissue dissociation, then addedto MEF media containing 10% FBS and plated (passage 0).

Retrovirus Infection.

The retroviral packaging vector pCL-ECO was transfected into 293T cellssimultaneously with pMXs vectors containing Oct4, Sox2, Klf4 or c-myccDNA(Addgene) using Fugene 6 (Roche) (Takahashi, K. & Yamanaka, S.Induction of pluripotent stem cells from mouse embryonic and adultfibroblast cultures by defined factors. Cell 126, 663-676 (2006)). At 24h, the media was changed, and at 48 h, the media was collected, filtered(0.45 μM), and frozen in aliquots at −80° C. Retrovirus was never thawedmore than once. To induce reprogramming, passage 3 Oct4-GFP,Rosa26-Glb1/neo MEFs49 (Blelloch, R., Venere, M., Yen, J. &Ramalho-Santos, M. Generation of induced pluripotent stem cells in theabsence of drug selection. Cell Stem Cell 1, 245-247 (2007)) were platedon gelatin-coated 12-well plates at 12,000 cells per well.Retrovirus-containing media was added 24 h later (day 0). Cells weretransfected with 16 nM microRNA inhibitors (Dharmacon, ThermoFisher,I-310106-04 for let-7 inhibitor, IN-001000-01-05 for control inhibitor).Cells in reprogramming assays were transfected on days 0 and 6 afterretroviral infection. Media was changed daily. Media was replaced withESC media+15% FBS+LIF on day 2, and ESC media+15% knockout serumreplacement (Invitrogen)+LIF on day 6. GFP colonies were counted on day10. Individual iPS cell colonies were picked and expanded for analysisbetween days 10 and 15.

Example 3

Additional Identified Useful ESCC miRNA Mimics. Identified During aScreen of Three Factors (Oct4, Sox2, KLF4+ microRNA Mimic).

GFP⁺ Colonies, Normalized to Mock

microRNA mimic exp. 1 exp. 2 average mmu-miR-302b 11.8 65.3 38.6mmu-miR-302 14 26.7 20.4 mmu-miR-302d 8.7 12.7 10.7 mmu-miR-294 3.2 169.6 mmu-miR-302c 2.2 14 8.1 mmu-miR-295 0 12.8 6.4 mmu-miR-93 1.6 10.86.2 mmu-miR-291-3p 6.4 0 3.2 mmu-miR-19a 0 5.2 2.6 mmu-miR-106a 0 4.52.3 mmu-miR-223 2.9 0.7 1.8 mmu-miR-291b-3p 2.1 0 1.1 mmu-miR-20b 1.1 00.6 mmu-miR-33 0 0 0

Example 4

Enhancers of reprogramming, defined by having GFP⁺ colony number greaterthan all the mocks in at least one screen and having at least a 2× foldaverage increase (52 mimics total). Identified during a screen of threefactors (Oct4, Sox2, KLF4⁺ microRNA mimic).

Generally, the reprogramming factors (in this case Oct4, Sox2, and Klf4via retrovirus) are added to the somatic cells (in this case Oct4-GFPMEFs) and after waiting 2 weeks, while changing media, two parametersare evaluated. Cells are also transfected with individual miRNA mimicsas already described in the patent. First, GFP+ colonies are counted. Ithas previously been shown that reactivation of the silenced Oct4-GFPtransgene in the MEFs is a very late marker of reprogramming, indicativeof a full pluripotent state. The cells are also stained and counted foralkaline phosphatase (AP) positive colonies. It has previously beenshown that AP activation is a very early step in the reprogrammingprocess, but is not as useful for indicating full reprogramming—ie, itmarks that the process has begun but not necessarily finished.

For the screen, which is depicted in Examples 4 and 5, wells treatedwith no miRNA—that is just Oct4, Sox2 and Klf4 retrovirus andtransfection reagent typically yield ˜0 to 4 GFP+ colonies. Usually, 16of these no miRNA mock wells are run per plate which means, perexperiment, each miRNA-including well is being compared to the averageof 16 no miRNA mock wells. The “fold change” then, is the number ofGFP+(or AP+) colonies from the microRNA-containing well divided by theaverage of the number of GFP+(or AP+) colonies of the 16 mock wells.Although many mock wells gave 0 colonies, the average of the 16 mockwells was always greater than zero and less then two. Thus, if one ofthe miRNA-containing wells had no GFP⁺ colonies, the normalized value isgoing to be 0. Take, for example, mir-291-3p which, over manyexperiments we have previously established as an enhancer ofreprogramming, but in one of the wells of the screen, gave a O-likely,more of an indicator that that particular well did not work great. Thusthe strongest candidates are those that yielded>2-3× average increasesin efficiency and indeed, most of the ESCCs fall into this category. Asfor the rest, they still have promising potential to be enhancers.

Number of GFP+ colonies divided by the average of the number of GFP+colonies in the mock group

microRNA mimic exp. 1 exp. 2 ave. sequence mmu-miR-302b 11.8 65.3 38.6UAAGUGCUUCCAUGUUUUAGUAG mmu-miR-302 14 26.7 20.3 UAAGUGCUUCCAUGUUUUGGUGAmmu-miR-495 27.2 0 13.6 AAACAAACAUGGUGCACUUCUU mmu-miR-26a 1.1 23.3 12.2UUCAAGUAAUCCAGGAUAGGCU mmu-miR-19a* 6 16 11 UAGUUUUGCAUAGUUGCACUACmmu-miR-302d 8.7 12.7 10.7 UAAGUGCUUCCAUGUUUGAGUGU mmu-miR-10b 1.1 18.79.9 UACCCUGUAGAACCGAAUUUGUG mmu-miR-294 3.2 16 9.6AAAGUGCUUCCCUUUUGUGUGU mmu-miR-302c 2.2 14 8.1 AAGUGCUUCCAUGUUUCAGUGGmmu-miR-183* 0 16 8 GUGAAUUACCGAAGGGCCAUAA mmu-miR-200a 0 16 8UAACACUGUCUGGUAACGAUGU mmu-miR-34c* 4.8 9.8 7.3 AAUCACUAACCACACAGCCAGGmmu-miR-293 1.6 12.8 7.2 AGUGCCGCAGAGUUUGUAGUGU mmu-miR-181b 0 14 7AACAUUCAUUGCUGUCGGUGGGU mmu-miR-151 0 13.3 6.7 CUAGACUGAGGCUCCUUGAGGmmu-miR-680 0 12.8 6.4 GGGCAUCUGCUGACAUGGGGG mmu-miR-295 0 12.8 6.4AAAGUGCUACUACUUUUGAGUCU mmu-miR-880 2 10.7 6.3 UACUCCAUCCUCUCUGAGUAGAmmu-miR-93 1.6 10.8 6.2 CAAAGUGCUGUUCGUGCAGGUAG mmu-miR-455-5p 6 5.3 5.7UAUGUGCCUUUGGACUACAUCG mmu-miR-144 8.7 2.6 5.7 UACAGUAUAGAUGAUGUACUmmu-miR-467d 5.3 5.6 5.5 UAAGUGCGCGCAUGUAUAUGCG mmu-miR-484 0 10.7 5.3UCAGGCUCAGUCCCCUCCCGAU mmu-miR-205 0 10.7 5.3 UCCUUCAUUCCACCGGAGUCUGmmu-miR-582-5p 10.2 0.4 5.3 UACAGUUGUUCAACCAGUUACU mmu-miR-290-3p 2 8 5AAAGUGCCGCCUAGUUUUAAGCCC mmu-miR-138* 2 8 5 CGGCUACUUCACAACACCAGGGmmu-miR-181d 8 1.9 4.9 AACAUUCAUUGUUGUCGGUGGGU mmu-miR-324-3p 7.3 2.24.8 CCACUGCCCCAGGUGCUGCU mmu-miR-877* 0 8 4 UGUCCUCUUCUCCCUCCUCCCAmmu-miR-23a 8 0 4 AUCACAUUGCCAGGGAUUUCC mmu-miR-379 0 8 4UGGUAGACUAUGGAACGUAGG mmu-miR-673 8 0 4 CUCACAGCUCUGGUCCUUGGAGmmu-miR-876-5p 4.8 2.6 3.7 UGGAUUUCUCUGUGAAUCACUA mmu-miR-291-3p 6.4 03.2 AAAGUGCUUCCACUUUGUGUGC mmu-miR-30d 3.2 3.1 3.1UGUAAACAUCCCCGACUGGAAG mmu-miR-421 5.8 0.4 3.1 AUCAACAGACAUUAAUUGGGCGCmmu-miR-879* 2.7 3.5 3.1 GCUUAUGGCUUCAAGCUUUCGG mmu-miR-542-3p 6 0 3UGUGACAGAUUGAUAACUGAAA mmu-miR-124* 1.6 4.1 2.9 CGUGUUCACAGCGGACCUUGAUmmu-miR-363 1.5 4.1 2.8 AAUUGCACGGUAUCCAUCUGUA mmu-miR-871 2.7 2.5 2.6UAUUCAGAUUAGUGCCAGUCAUG mmu-miR-19a 0 5.2 2.6 UGUGCAAAUCUAUGCAAAACUGAmmu-miR-16* 2.7 2.2 2.4 CCAGUAUUGACUGUGCUGCUGA mmu-miR-873 1.5 3.3 2.4GCAGGAACUUGUGAGUCUCCU mmu-miR-199b 0 4.6 2.3 CCCAGUGUUUAGACUACCUGUUCmmu-miR-106a 0 4.5 2.2 CAAAGUGCUAACAGUGCAGGUAG mmu-miR-181b 4.3 0 2.2AACAUUCAUUGCUGUCGGUGGGU mmu-miR-200a* 4.3 0 2.1 CAUCUUACCGGACAGUGCUGGAmmu-miR-431* 4.3 0 2.1 CAGGUCGUCUUGCAGGGCUUCU mmu-miR-689 1.6 2.6 2.1CGUCCCCGCUCGGCGGGGUCC mmu-miR-721 1.6 2.6 2.1 CAGUGCAAUUAAAAGGGGGAA

Example 5

Inhibitors of reprogramming, defined by having AP+ colony number lessthan all of the mocks in at least one screen and having at least a 40%average reduction (114 mimics total). Identified during a screen ofthree factors (Oct4, Sox2, KLF4+microRNA mimic).

AP+ Colonies, Normalized to Mock:

Number of AP+ colonies divided by the average of the number of AP+colonies in the mock group

microRNA mimic exp. 1 exp. 2 Ave. sequence mmu-miR-744* 0 0.14 0.07CUGUUGCCACUAACCUCAACCU mmu-miR-423-5p 0 0.27 0.14UGAGGGGCAGAGAGCGAGACUUU mmu-miR-669c 0 0.28 0.14 AUAGUUGUGUGUGGAUGUGUGUmmu-let-7c 0 0.28 0.14 UGAGGUAGUAGGUUGUAUGGUU mmu-miR-466h 0 0.28 0.14UGUGUGCAUGUGCUUGUGUGUA mmu-miR-654-3p 0 0.35 0.18 UAUGUCUGCUGACCAUCACCUUmmu-miR-470* 0.22 0.15 0.19 AACCAGUACCUUUCUGAGAAGA mmu-miR-24 0 0.41 0.2UGGCUCAGUUCAGCAGGAACAG mmu-miR-182 0.11 0.3 0.21UUUGGCAAUGGUAGAACUCACACCG mmu-miR-335 0 0.41 0.21UCAAGAGCAAUAACGAAAAAUGU mmu-miR-181c 0 0.41 0.21 AACAUUCAACCUGUCGGUGAGUmmu-miR-330 0 0.41 0.21 GCAAAGCACAGGGCCUGCAGAGA mmu-miR-134 0 0.41 0.21UGUGACUGGUUGACCAGAGGGG mmu-miR-675-3p 0.29 0.19 0.24CUGUAUGCCCUAACCGCUCAGU mmu-miR-218 0.29 0.19 0.24 UUGUGCUUGAUCUAACCAUGUmmu-let-7f 0 0.49 0.25 UGAGGUAGUAGAUUGUAUAGUU mmu-miR-491 0.14 0.38 0.26AGUGGGGAACCCUUCCAUGAGG mmu-miR-466g 0.26 0.27 0.26 AUACAGACACAUGCACACACAmmu-miR-465c-3p 0.26 0.27 0.26 GAUCAGGGCCUUUCUAAGUAGA mmu-miR-202 0 0.540.27 AGAGGUAUAGCGCAUGGGAAGA mmu-miR-681 0 0.54 0.27CAGCCUCGCUGGCAGGCAGCU mmu-miR-877 0 0.54 0.27 GUAGAGGAGAUGGCGCAGGGmmu-miR-875-5p 0 0.54 0.27 UAUACCUCAGUUUUAUCAGGUG mmu-miR-712 0 0.560.28 CUCCUUCACCCGGGCGGUACC mmu-miR-297 0.22 0.35 0.29AUGUAUGUGUGCAUGUGCAUGU mmu-let-7d 0.44 0.15 0.29 AGAGGUAGUAGGUUGCAUAGUUmmu-miR-142-3p 0.11 0.49 0.3 UGUAGUGUUUCCUACUUUAUGGA mmu-miR-328 0.480.14 0.31 CUGGCCCUCUCUGCCCUUCCGU mmu-miR-485-5p 0.48 0.14 0.31AGAGGCUGGCCGUGAUGAAUUC mmu-miR-122a 0.22 0.42 0.32UGGAGUGUGACAAUGGUGUUUG mmu-miR-877* 0.26 0.41 0.33UGUCCUCUUCUCCCUCCUCCCA mmu-miR-135a 0.14 0.56 0.35UAUGGCUUUUUAUUCCUAUGUGA mmu-miR-674-3p 0.14 0.56 0.35CACAGCUCCCAUCUCAGAACAA mmu-miR-497 0.14 0.56 0.35 CAGCAGCACACUGUGGUUUGUAmmu-miR-7b 0.33 0.42 0.38 UGGAAGACUUGUGAUUUUGUUGU mmu-miR-30b* 0.26 0.490.38 CUGGGAUGUGGAUGUUUACGUC mmu-miR-34b 0.33 0.45 0.39AGGCAGUGUAAUUAGCUGAUUGU mmu-miR-466e-5p 0 0.79 0.4GAUGUGUGUGUACAUGUACAUA mmu-miR-193b 0.26 0.54 0.4 AACUGGCCCACAAAGUCCCGCUmmu-miR-883a-5p 0.26 0.54 0.4 UGCUGAGAGAAGUAGCAGUUAC mmu-let-7i* 0.170.64 0.41 CUGCGCAAGCUACUGCCUUGCU mmu-miR-342 0.11 0.71 0.41UCUCACACAGAAAUCGCACCCGU mmu-miR-140* 0.11 0.71 0.41UACCACAGGGUAGAACCACGG mmu-miR-24-2* 0.26 0.56 0.41GUGCCUACUGAGCUGAAACAGU mmu-miR-195 0.12 0.71 0.42 UAGCAGCACAGAAAUAUUGGCmmu-miR-297a 0.12 0.71 0.42 AUGUAUGUGUGCAUGUGCAUGU mmu-miR-344 0.12 0.710.42 UGAUCUAGCCAAAGCCUGACUGU mmu-miR-18 0.77 0.07 0.42UAAGGUGCAUCUAGUGCAGAUAG mmu-miR-93* 0.35 0.5 0.42 ACUGCUGAGCUAGCACUUCCCGmmu-miR-297 0.66 0.21 0.43 AUGUAUGUGUGCAUGUGCAUGU mmu-miR-16 0.24 0.630.44 UAGCAGCACGUAAAUAUUGGCG mmu-miR-380-5p 0.52 0.36 0.44AUGGUUGACCAUAGAACAUGCG mmu-miR-672 0.17 0.71 0.44UGAGGUUGGUGUACUGUGUGUGA mmu-miR-431 0.33 0.57 0.45 UGUCUUGCAGGCCGUCAUGCAmmu-miR-715 0.14 0.75 0.45 CUCCGUGCACACCCCCGCGUG mmu-miR-669a 0.14 0.750.45 AGUUGUGUGUGCAUGUUCAUGU mmu-miR-103 0.48 0.41 0.45AGCAGCAUUGUACAGGGCUAUGA mmu-miR-124* 0.12 0.79 0.46CGUGUUCACAGCGGACCUUGAU mmu-miR-15b 0.12 0.79 0.46 UAGCAGCACAUCAUGGUUUACAmmu-miR-450b* 0.12 0.79 0.46 AUUGGGAACAUUUUGCAUGCAU mmu-miR-882 0.350.57 0.46 AGGAGAGAGUUAGCGCAUUAGU mmu-miR-686 0.52 0.41 0.46AUUGCUUCCCAGACGGUGAAGA mmu-miR-222 0.22 0.71 0.46 AGCUACAUCUGGCUACUGGGUmmu-miR-684 0 0.94 0.47 AGUUUUCCCUUCAAGUCAA mmu-miR-450b 0.24 0.71 0.47UUUUGCAGUAUGUUCCUGAAUA mmu-miR-582-3p 0.17 0.78 0.48CCUGUUGAACAACUGAACCCAA mmu-miR-135b 0.17 0.78 0.48UAUGGCUUUUCAUUCCUAUGUGA mmu-miR-493 0.26 0.7 0.48UGAAGGUCCUACUGUGUGCCAGG mmu-miR-546 0.97 0 0.48 AUGGUGGCACGGAGUCmmu-miR-708 0.22 0.75 0.49 AAGGAGCUUACAAUCUAGCUGGG mmu-miR-433-3p 0.120.87 0.49 AUCAUGAUGGGCUCCUCGGUGU mmu-miR-494 0.22 0.78 0.5UGAAACAUACACGGGAAACCUC mmu-miR-203 0.52 0.5 0.51 GUGAAAUGUUUAGGACCACUAGmmu-miR-9 0.52 0.5 0.51 UCUUUGGUUAUCUAGCUGUAUGA mmu-miR-574-5p 0.39 0.630.51 UGAGUGUGUGUGUGUGAGUGUGU mmu-miR-376c 0.17 0.85 0.51AACAUAGAGGAAAUUUCACCU mmu-miR-433-5p 0 1.03 0.51 UACGGUGAGCCUGUCAUUAUUCmmu-miR-181a-2* 0 1.03 0.51 ACCGACCGUUGACUGUACCUUG mmu-miR-218-2* 0.260.77 0.52 CAUGGUUCUGUCAAGCACCGCG mmu-miR-196a 0.33 0.71 0.52UAGGUAGUUUCAUGUUGUUGGG mmu-miR-542-5p 0.33 0.71 0.52CUCGGGGAUCAUCAUGUCACGA mmu-miR-7 0.55 0.49 0.52 UGGAAGACUAGUGAUUUUGUUGUmmu-miR-743b-5p 0.13 0.91 0.52 UGUUCAGACUGGUGUCCAUCA mmu-miR-377 0.770.27 0.52 AUCACACAAAGGCAACUUUUGU mmu-miR-683 0.77 0.27 0.52CCUGCUGUAAGCUGUGUCCUC mmu-miR-675-5p 0.86 0.19 0.53UGGUGCGGAAAGGGCCCACAGU mmu-miR-598 0.52 0.54 0.53 UACGUCAUCGUCGUCAUCGUUAmmu-miR-15b* 0.12 0.95 0.53 CGAAUCAUUAUUUGCUGCUCUA mmu-miR-9 0.66 0.420.54 UCUUUGGUUAUCUAGCUGUAUGA mmu-miR-450a-3p 0.33 0.75 0.54AUUGGGGAUGCUUUGCAUUCAU mmu-miR-449b 0.33 0.75 0.54 AGGCAGUGUUGUUAGCUGGCmmu-miR-707 0.14 0.94 0.54 CAGUCAUGCCGCUUGCCUACG mmu-miR-335-3p 0.520.56 0.54 UUUUUCAUUAUUGCUCCUGACC mmu-miR-147 0.39 0.7 0.55GUGUGCGGAAAUGCUUCUGCUA mmu-miR-466c-5p 0.26 0.84 0.55GAUGUGUGUGUGCAUGUACAUA mmu-miR-16 0.24 0.87 0.55 UAGCAGCACGUAAAUAUUGGCGmmu-miR-127 0.77 0.35 0.56 UCGGAUCCGUCUGAGCUUGGCU mmu-miR-673-3p 0.70.43 0.56 UCCGGGGCUGAGUUCUGUGCACC mmu-miR-466b-5p 0.22 0.91 0.56GAUGUGUGUGUACAUGUACAUG mmu-miR-27a* 0.22 0.91 0.56AGGGCUUAGCUGCUUGUGAGCA mmu-miR-1 0.35 0.78 0.57 UGGAAUGUAAAGAAGUAUGUAUmmu-miR-201 0.22 0.92 0.57 UACUCAGUAAGGCAUUGUUCUU mmu-miR-376b 0.22 0.920.57 AUCAUAGAGGAACAUCCACUU mmu-miR-187 0.12 1.03 0.57UCGUGUCUUGUGUUGCAGCCGG mmu-miR-299 0.12 1.03 0.57 UGGUUUACCGUCCCACAUACAUmmu-miR-299 0.39 0.77 0.58 UAUGUGGGACGGUAAACCGCUU mmu-miR-574-3p 0.390.77 0.58 CACGCUCAUGCACACACCCACA mmu-miR-193* 0.39 0.77 0.58UGGGUCUUUGCGGGCAAGAUGA mmu-miR-679 0.48 0.69 0.59 GGACUGUGAGGUGACUCUUGGUmmu-miR-540-5p 0.26 0.91 0.59 CAAGGGUCACCCUCUGACUCUGU mmu-miR-466a-5p0.26 0.91 0.59 UAUGUGUGUGUACAUGUACAUA mmu-miR-470 0.33 0.85 0.59UUCUUGGACUGGCACUGGUGAGU mmu-miR-1224 0.77 0.41 0.59GUGAGGACUGGGGAGGUGGAG mmu-miR-191 0.55 0.64 0.59 CAACGGAAUCCCAAAAGCAGCUG

Example 6

miRNA sequences of FIG. 20.

Accession MicroRNA No. Sequence hsa-miR- MIMAT0000263CCCAGUGUUUAGACUAUCU 199b-5p GUUC hsa-let-7a MIMAT0000062UGAGGUAGUAGGUUGUAUA GUU hsa-let-7b MIMAT0000063 UGAGGUAGUAGGUUGUGUG GUUhsa-let-7c MIMAT0000064 UGAGGUAGUAGGUUGUAUG GUU hsa-let-7d MIMAT0000065AGAGGUAGUAGGUUGCAUA GUU hsa-let-7e MIMAT0000066 UGAGGUAGGAGGUUGUAUA GUUhsa-let-7f MIMAT0000067 UGAGGUAGUAGAUUGUAUA GUU hsa-let-7g MIMAT0000414UGAGGUAGUAGUUUGUACA GUU hsa-let-7i MIMAT0000415 UGAGGUAGUAGUUUGUGCU GUUhsa-miR-100 MIMAT0000098 AACCCGUAGAUCCGAACUU GUG hsa-miR-100MIMAT0000098 AACCCGUAGAUCCGAACUU GUG hsa-miR-122 MIMAT0000421UGGAGUGUGACAAUGGUGU UUG hsa-miR-127- MIMAT0000446 UCGGAUCCGUCUGAGCUUG 3pGCU hsa-miR-128 MIMAT0000424 UCACAGUGAACCGGUCUCU UU hsa-miR-129-MIMAT0000242 CUUUUUGCGGUCUGGGCUU 5p GC hsa-miR-134 MIMAT0000447UGUGACUGGUUGACCAGAG GGG hsa-miR-140- MIMAT0000431 CAGUGGUUUUACCCUAUGG 5pUAG hsa-miR-145 MIMAT0000437 GUCCAGUUUUCCCAGGAAU CCCU hsa-miR-149MIMAT0000450 UCUGGCUCCGUGUCUUCAC UCCC hsa-miR-18a MIMAT0000072UAAGGUGCAUCUAGUGCAG AUAG hsa-miR-18b MIMAT0001412 UAAGGUGCAUCUAGUGCAGUUAG hsa-miR-193a- MIMAT0000459 AACUGGCCUACAAAGUCCC 3p AGU hsa-miR-199a-MIMAT0000231 CCCAGUGUUCAGACUACCU 5p GUUC hsa-miR-216a MIMAT0000273UAAUCUCAGCUGGCAACUG UGA hsa-miR-216b MIMAT0004959 AAAUCUCUGCAGGCAAAUGUGA hsa-miR-218 MIMAT0000275 UUGUGCUUGAUCUAACCAU GU hsa-miR-26aMIMAT0000082 UUCAAGUAAUCCAGGAUAG GCU hsa-miR-31 MIMAT0000089AGGCAAGAUGCUGGCAUAG CU hsa-miR-345 MIMAT0000772 GCUGACUCCUAGUCCAGGG CUChsa-miR-34c- MIMAT0000686 AGGCAGUGUAGUUAGCUGA 5p UUGC hsa-miR-362-MIMAT0000705 AAUCCUUGGAACCUAGGUG 5p UGAGU hsa-miR-378 MIMAT0000732ACUGGACUUGGAGUCAGAA GG hsa-miR-384 MIMAT0001075 AUUCCUAGAAAUUGUUCAU Ahsa-miR-409- MIMAT0001639 GAAUGUUGCUCGGUGAACC 3p CCU hsa-miR-450aMIMAT0001545 UUUUGCGAUGUGUUCCUAA UAU hsa-miR-450b- MIMAT0004909UUUUGCAAUAUGUUCCUGA 5p AUA hsa-miR-452 MIMAT0001635 AACUGUUUGCAGAGGAAACUGA hsa-miR-98 MIMAT0000096 UGAGGUAGUAAGUUGUAUU GUU hsa-miR-99aMIMAT0000097 AACCCGUAGAUCCGAUCUU GUG hsa-miR-99b MIMAT0000689CACCCGUAGAACCGACCUU GCG mmu-let-7a MIMAT0000521 UGAGGUAGUAGGUUGUAUA GUUmmu-let-7b MIMAT0000522 UGAGGUAGUAGGUUGUGUG GUU mmu-let-7c MIMAT0000523UGAGGUAGUAGGUUGUAUG GUU mmu-let-7d MIMAT0000383 AGAGGUAGUAGGUUGCAUA GUUmmu-let-7e MIMAT0000524 UGAGGUAGGAGGUUGUAUA GUU mmu-let-7f MIMAT0000525UGAGGUAGUAGAUUGUAUA GUU mmu-let-7g MIMAT0000121 UGAGGUAGUAGUUUGUACA GUUmmu-let-7i MIMAT0000122 UGAGGUAGUAGUUUGUGCU GUU mmu-miR-100 MIMAT0000655AACCCGUAGAUCCGAACUU GUG mmu-miR-100 MIMAT0000655 AACCCGUAGAUCCGAACUU GUGmmu-miR-122 MIMAT0000246 UGGAGUGUGACAAUGGUGU UUG mmu-miR-127MIMAT0000139 UCGGAUCCGUCUGAGCUUG GCU mmu-miR-128 MIMAT0000140UCACAGUGAACCGGUCUCU UU mmu-miR-129- MIMAT0000209 CUUUUUGCGGUCUGGGCUU 5pGC mmu-miR-134 MIMAT0000146 UGUGACUGGUUGACCAGAG GGG mmu-miR-140MIMAT0000151 CAGUGGUUUUACCCUAUGG UAG mmu-miR-145 MIMAT0000157GUCCAGUUUUCCCAGGAAU CCCU mmu-miR-149 MIMAT0000159 UCUGGCUCCGUGUCUUCACUCCC mmu-miR-18a MIMAT0000528 UAAGGUGCAUCUAGUGCAG AUAG mmu-miR-18bMIMAT0004858 UAAGGUGCAUCUAGUGCUG UUAG mmu-miR-193 MIMAT0000223AACUGGCCUACAAAGUCCC AGU mmu-miR-199a- MIMAT0000229 CCCAGUGUUCAGACUACCU5p GUUC mmu-miR-199b MIMAT0004667 ACAGUAGUCUGCACAUUGG UUA mmu-miR-216aMIMAT0000662 UAAUCUCAGCUGGCAACUG UGA mmu-miR-216b MIMAT0003729AAAUCUCUGCAGGCAAAUG UGA mmu-miR-218 MIMAT0000663 UUGUGCUUGAUCUAACCAU GUmmu-miR-26a MIMAT0000533 UUCAAGUAAUCCAGGAUAG GCU mmu-miR-31 MIMAT0000538AGGCAAGAUGCUGGCAUAG CUG mmu-miR-345 MIMAT0000595 GCUGACCCCUAGUCCAGUG CUUmmu-miR-34c MIMAT0000381 AGGCAGUGUAGUUAGCUGA UUGC mmu-miR-362-MIMAT0000706 AAUCCUUGGAACCUAGGUG 5p UGAAU mmu-miR-378 MIMAT0003151ACUGGACUUGGAGUCAGAA (old mmu- GG miR-422b) mmu-miR-384- MIMAT0001076AUUCCUAGAAAUUGUUCAC 3p AAU mmu-miR-409- MIMAT0001090 GAAUGUUGCUCGGUGAACC3p CCU mmu-miR-450a- MIMAT0001546 UUUUGCGAUGUGUUCCUAA 5p UAUmmu-miR-450b- MIMAT0003511 UUUUGCAGUAUGUUCCUGA 5p AUA mmu-miR-452MIMAT0001637 UGUUUGCAGAGGAAACUGA GAC mmu-miR-464 MIMAT0002105UACCAAGUUUAUUCUGUGA GAUA mmu-miR-465a- MIMAT0002106 UAUUUAGAAUGGCACUGAU5p GUGA mmu-miR-465b- MIMAT0004871 UAUUUAGAAUGGUGCUGAU 5p CUGmmu-miR-465c- MIMAT0004873 UAUUUAGAAUGGCGCUGAU 5p CUG mmu-miR-468UAUGACUGAUGUGCGUGUG UCUG mmu-miR-98 MIMAT0000545 UGAGGUAGUAAGUUGUAUU GUUmmu-miR-99a MIMAT0000131 AACCCGUAGAUCCGAUCUU GUG mmu-miR-99bMIMAT0000132 CACCCGUAGAACCGACCUU GCG old mmu-miR- CUGGACUUGGAGUCAGAAG422b GC

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein, including but not limited to those listedbelow, are hereby incorporated by reference in their entirety for allpurposes.

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1-40. (canceled)
 41. A method of inducing pluripotency in an isolatedcell comprising: (a) introducing a vector(s) encoding the reprogrammingfactors Oct-4, Sox2, KLF-4 (OSK) into the cell; (b) introducing aninhibitor of a member of the let-7 family, wherein the member of thelet-7 family has the seed sequence GAGGUAG; and (c) culturing the cellfrom step (b) to produce a pluripotent cell, wherein the inhibitor andthe expression of the reprogramming factors results in an enhancedproduction of pluripotent cells when compared to introduction of thereprogramming factors in the absence of the inhibitor.
 42. The method ofclaim 41, wherein the inhibitor inhibits at least two let-7 familymembers simultaneously.
 43. The method of claim 41, wherein theinhibitor comprises a sequence complementary to the seed sequenceGAGGUAG.
 44. The method of claim 41, further comprising introducing aphysiologically relevant miRNA into the cell, wherein the relevant miRNAcontains the seed sequence AAGUGCU (SEQ ID NO:15), AAAGUGC (SEQ IDNO:16) or AAGUGC.
 45. The method of claim 44, wherein the relevant miRNAis a member of the embryonic stem cell cycle (ESCC) regulating miR-290cluster.
 46. The method of claim 44, wherein the relevant miRNA is amember of the embryonic stem cell cycle (ESCC) regulating 302 cluster,17-92 cluster, 106a, and 370 family of human miRNAs.
 47. The method ofclaim 44, wherein the relevant miRNA is a human or mouse miRNA.
 48. Themethod of claim 41, wherein the cell is a human cell.
 49. The method ofclaim 48, wherein the human cell is a somatic cell.
 50. The method ofclaim 44, wherein the relevant miRNA is (i) 80% or more identical to oneof miR-291-3p (SEQ ID NO:1), miR-294 (SEQ ID NO:2), miR-295 (SEQ IDNO:3), miR-302d (SEQ ID NO:4), miR-292-3p (SEQ ID NO:5), hsa-mir-302a(SEQ ID NO:8), hsa-miR-302b (SEQ ID NO:9), hsa-miR-302c (SEQ ID NO:10),hsa-miR-302d (SEQ ID NO:11), hsa-miR-372 (SEQ ID NO:13), hsa-miR-373(SEQ ID NO:14), hsa-miR-17 (SEQ ID NO:17), hsa-miR-20a (SEQ ID NO:18),hsa-miR-20b (SEQ ID NO:19), hsa-miR-93 (SEQ ID NO:20), hsa-miR-106a (SEQID NO:21), or hsa-miR-106b (SEQ ID NO:22); and (ii) contains the seedsequence AAGUGCU (SEQ ID NO: 15), AAAGUGC (SEQ ID NO:16) or AAGUGC. 51.The method of claim 44, wherein the miR-290 cluster member is one ofmiR-291-3p (SEQ ID NO:1), miR-294 (SEQ ID NO:2) miR-295 (SEQ ID NO:3),miR-302d (SEQ ID NO:4), miR-292-3p (SEQ ID NO:5), hsa-mir-302a (SEQ IDNO:8), hsa-miR-302b (SEQ ID NO:9), hsa-miR-302c (SEQ ID NO:10),hsa-miR-302d (SEQ ID NO:11), hsa-miR371-5p (SEQ ID NO:12), hsa-miR-372(SEQ ID NO:13), or hsa-miR-373 (SEQ ID NO:14).
 52. The method of claim44, wherein the miRNA that contains the seed sequence AAGUGCU (SEQ IDNO:15), AAAGUGC (SEQ ID NO:16) or AAGUGC is a miRNA of Example 3 orExample 4 or a human ortholog thereof.
 53. The method of claim 41,wherein the pluripotent cell is introduced into a mammalian host. 54.The method of claim 53, wherein the cell is autologous to the host. 55.The method of claim 54, wherein the host has a degenerative condition.